Comparison of Protein Hydrolysis Catalyzed by Bovine, Porcine, and

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Comparison of protein hydrolysis catalysed by bovine, porcine or human trypsins Yuxi Deng, Harry Gruppen, and Peter A. Wierenga J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00679 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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Journal of Agricultural and Food Chemistry

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Comparison of protein hydrolysis catalysed by bovine,

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porcine and human trypsins

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Yuxi Denga, Harry Gruppena and Peter A. Wierengaa*

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a

Laboratory of Food Chemistry, Wageningen University, P.O.Bus 17, 6708 AA Wageningen , The Netherlands

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*Corresponding author:

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Telephone: +31 317483786

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Email: [email protected]

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Email:

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Yuxi Deng: [email protected]

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Harry Gruppen: [email protected]

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1 ACS Paragon Plus Environment


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Abstract (150 words):

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Based on trypsin specificity (lysine, arginine) trypsins from different sources are expected to

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hydrolyse a given protein to the same theoretical maximum degree of hydrolysis (DHmax,theo).

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This is in contrast with experiments. This study aims to reveal if the difference in

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experimental DHmax (DHmax,exp) by bovine, porcine or human trypsins is due to their

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secondary specificity, using α-lactalbumin and β-casein. Peptide analysis showed that from

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all cleavage sites ~78% were efficiently hydrolysed by porcine trypsin, and ~47% and ~53%

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were efficiently hydrolysed by bovine and human trypsins, respectively. These differences

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were explained by the enzyme secondary specificity, i.e. the sensitivity to the amino acids

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around the cleavage sites. The DHmax prediction based on secondary specificity was 4 times

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closer to DHmax,exp than the prediction based on specificity (DHmax,theo). Proposed preliminary

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relations between binding sites and trypsin secondary specificity allow the DHmax,exp

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estimation of tryptic hydrolysis of other proteins.

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Key words: LC-MS, peptide release kinetics, tryptic hydrolysis, protein digestibility,

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secondary specificity


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Introduction

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The in vivo protein digestion in the intestines is often simulated by the in vitro hydrolysis

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using pancreatin, in which the main proteases are trypsin and chymotrypsin. Trypsins

34

specifically hydrolyse peptide bonds on the carboxylic side of lysine and arginine. In principle,

35

when the specificity of the enzyme is known, as well as the amino acid (AA) sequence of the

36

protein, the theoretical maximum degree of hydrolysis (DHmax,theo) can be calculated. In the

37

case of tryptic hydrolysis of α-lactalbumin, the DHmax,theo is 10.7 % (Table 2). When this

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protein was hydrolysed by porcine trypsin, the experimental maximum degree of hydrolysis

39

(DHmax,exp) (~10.7 %) was indeed similar to DHmax,theo 1. Using bovine trypsin, surprisingly, the

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DHmax,exp was only ~3.6 % 1. Peptide analysis showed that the lower DHmax,exp reached by

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bovine trypsin was because 8 out of 14 cleavage sites (CSs) on the protein were hydrolysed

42

by the enzyme only at a low rate 2. These low hydrolysis rate constants were proposed to be

43

due to the hindrance of bovine trypsin by the charged AAs surrounding the CSs 2 (Table 1).

44

The differences in hydrolysis rate constants of CSs in a protein are quantitatively described

45

by enzyme “selectivity” 3. The current study aims to investigate the hydrolysis by bovine,

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porcine and human trypsins, and to study if the differences in DHmax,exp could be explained

47

based on the enzyme selectivity. A detailed understanding of the differences in enzyme

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selectivity of the trypsins will allow a better understanding of the reported in vitro

49

digestibility of a protein.

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In commercial trypsins extracted from bovine and porcine pancreas, two variants of

51

trypsin, i.e. cationic (trypsin-1) and anionic (trypsin-2) trypsins, are present. The trypsin-1 is

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the dominant variant. Human trypsin consists of three variants of trypsin. The content of

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trypsin-1 (cationic), trypsin-2 (anionic) and trypsin-3 (mesotrypsin) were found to be 60 %,

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30 % and 10 %, respectively 4. Unless stated otherwise, in this study, bovine and porcine 3 ACS Paragon Plus Environment


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trypsins are further referred to the trypsin-1, and human trypsin is further referred to the

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trypsin-2. Using bovine trypsin, the experimental maximum degree of hydrolysis (DHmax,exp)

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of α-lactalbumin, β-casein and β-lactoglobulin were reported to be 5.1 % 2, 4.3 % 2 and 8.0 %

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2, 5

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degree of hydrolysis (DHmax,theo) of each protein. The peptide analysis of α-lactalbumin

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hydrolysates showed that 2 out of 13 CSs were not hydrolysed 2. For the hydrolysed CSs, the

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hydrolysis rate constants of bovine trypsin towards the individual CSs varied from 4.7·10-5 to

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3.0·10-3 s-1·mg-1enzyme, which were equivalent to selectivity values (relative hydrolysis rate

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constants) ranging from 0.4 to 27.6 % 2. The 2 CSs that were not hydrolysed were referred to

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as the missed cleavages. The occurrence of missed cleavages was also shown in bovine

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tryptic hydrolysates of human serum albumin hydrolysates, where ~100 peptides with

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missed CSs were identified 6. The occurrence of missed cleavages in bovine tryptic hydrolysis

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has been attributed to the presence of charged AAs surrounding the CSs

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protease binds to a substrate, part of the enzyme (the subsite of the enzyme, S4-S4’)

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interacts with part of the substrate AA sequence (the binding site of the CS, P4-P4’) 9. The

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P4-P4’ positions are numbered from the N- to C-terminal sides of the CS, and P1 identifies

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the AA for which the enzyme is specific 9. Recently, this concept was applied to describe

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bovine trypsin selectivity towards CSs in α-lactalbumin, β-casein and β-lactoglobulin 2. To

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obtain a better understanding of bovine tryptic hydrolysis, the relation between

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experimental selectivity and molecular properties of the binding site positions was

75

investigated 2. Based on the relation, CSs could be differentiated by the charge of AAs on the

76

P2 and P2’ positons of the binding site sequence into 3 categories (Table 1). The 3 categories

77

were CS that were 1) hydrolysed efficiently, when neutral AAs are on the two positions (high

78

selectivity sites); 2) hydrolysed with an intermediate hydrolysis rate, when a charged AA is

, respectively. These values were only 48 %, 60 % and 75 % of the theoretical maximum


7-8

. When a

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on one of the two positions (intermediate selectivity sites); 3) hydrolysed slowly, when

80

charged AAs are on both positions (low/zero selectivity sites) 2. The type of binding site

81

sequences of CSs in the 3 categories was taken as a set of rules to predict the DHmax,exp of

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proteins (Table 1). Using these rules (based on selectivity), the predicted maximum degree

83

of hydrolysis was found to be ~5 times closer to the DHmax,exp than the DHmax,theo (the

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prediction based on specificity) 2.

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When comparing trypsin from different sources, significant differences have been found

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in the reached DHmax,exp and the hydrolysate composition during hydrolysis. For example, the

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DHmax,exp of porcine tryptic hydrolysis of α-lactalbumin was equal to the DHmax,theo 1.

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Moreover, in a porcine tryptic hydrolysate of human serum albumin, the number of unique

89

peptides that contained missed CSs was 60 % lower than in a bovine tryptic hydrolysate 6.

90

Based on these data, porcine trypsin is not expected to be largely influenced by the

91

molecular properties of the binding site sequences. Different peptide profiles were also

92

found between hydrolysates of β-lactoglobulin taken after 6 h of hydrolysis by bovine or

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ovine trypsins 10. These observations indicated that the reported in vitro protein digestibility

94

and the hydrolysate composition of one substrate protein determined by hydrolysis using

95

different trypsins could have various outcome.

96

In in vitro protein digestion models often bovine and porcine trypsins were used, also

97

when the aim was to simulate human digestive systems. A previous study from our group

98

showed that the bovine trypsin secondary specificity limited the DHmax,exp of α-lactalbumin,

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β-casein and β-lactoglobulin 2. In another study, we found that the DHmax,exp of α-lactalbumin

100

by porcine trypsin was higher than by bovine trypsin. In this study, the aim is to identify

101

differences in secondary specificity between the trypsins from different sources that result in

102

different DHmax,exp values. For this, the bovine, porcine or human tryptic hydrolysis of 5 ACS Paragon Plus Environment


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individual CSs in two proteins (α-lactalbumin and β-casein) was studied. The rate constants

104

with which the three trypsins hydrolyse CSs in the two proteins (selectivity) were

105

determined. The selectivity was linked to the molecular properties of binding site sequences,

106

to identify the secondary specificity of each trypsin.

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Materials and methods

108

Materials

109

α-Lactalbumin (α-LA) was obtained from Davisco Foods International Inc. (Le Sueur, MN,

110

USA). Based on results from circular dichroism, ~72 % of the α-LA was in apo form and the

111

rest was in holo form 1. β-Casein (β-cas, C6905), bovine trypsin (BT, T1426), porcine trypsin

112

(PT, T0303) and aprotinin (A6279) were all purchased from Sigma-Aldrich (St.Louis, MO,

113

USA). Recombinant human trypsin-2 (further referred to as human trypsin, abbreviated as

114

HT) was purchased from Shanghai Yaxin Biotechnology Co., Ltd (Shanghai, China). The

115

molecular properties, including the protein content and purity, of the two proteins, i.e. α-LA

116

and β-cas, and the three trypsins, i.e. BT, PT and HT, were listed in Table 2. To check the

117

composition of the enzymes, all three trypsins were analysed using ultra-performance liquid

118

chromatography coupled with mass spectrometry (UPLC-MS). There was no chymotrypsin

119

peak found in the chromatograms of any trypsins. The activities of bovine, porcine and

120

human trypsins were specified to be ≥10,000, 13,000-20,000 and ≥7500 benzoyl-L-arginine-

121

ethyl-ester units/mg protein by the manufacturers, respectively. Aprotinin was used to

122

inhibit each trypsin hydrolysis, as described below. Aprotinin present in 0.9 % sodium

123

chloride and 0.9 % benzylalcohol solution, was determined to have a concentration of 2.3

124

mg/mL based on the peak area at 214 nm (A214) using RP-UPLC-UV 2. All other chemicals

125

were of analytical grade and purchased from Sigma or Merck.


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Methods

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Enzymatic hydrolysis of the proteins

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The enzymatic hydrolysis of proteins was performed as described previously 3. The α-LA

129

or β-cas solutions (1 % [w powder/v], 10 mL in Millipore) were adjusted to pH 8.0 and

130

equilibrated for 0.5 h at 37 °C in a pH-stat device (Metrohm, Herisau, Switzerland). Trypsin

131

solution (100 µL, 10 mg/mL in Millipore) was added to the equilibrated protein solution to

132

reach an enzyme to substrate ratio of 1:100 [w/w]. The hydrolysis was performed at 37 °C

133

for 3 h using 0.2 M NaOH to keep the pH constant. The degree of hydrolysis (DHstat) was

134

calculated using equation 1 11,

135

(1) [%] = × × × ×

136

where Vb [mL] is the volume of NaOH added; Nb [mol/L] is the normality of NaOH; α is the

137

average degree of dissociation of the α-NH groups (1/α=1.3 at 37 °C and pH 8) 12; mp [g] is

138

the mass of protein in the solution (taking into account the protein content in the powder

139

based on Dumas results); htot [mmol/g] is the total number of millimoles of peptide bonds

140

per gram of protein substrate (Table 2). For each hydrolysis, at set DH values, 200 μL sample

141

was taken. The DH at which samples were taken depended on the DHmax,exp reached after 3 h

142

of hydrolysis. During α-LA hydrolysis, samples were taken at DH 0, 1.5, 3, 4.5, 6, 7.5, 9 and

143

10 % for porcine tryptic hydrolysis and at DH 0, 1, 2, 3, 4, 5 and 6.3 % for human trypsin

144

hydrolysis. During β-cas hydrolysis, samples were taken at DH 0, 1.5, 3, 4.5, 6, 6.7 % for

145

porcine tryptic hydrolysis and at DH 0, 1, 2, 3, 4, 5 and 5.2 % for human tryptic hydrolysis. In

146

a previous study, the same experiments were performed using bovine trypsin 2. In that study,

147

samples were taken at DH 0, 1, 2, 3, 4 and 4.7 % for α-LA and at DH 0, 1, 2, 3, 4 and 4.3 % for

148

β-cas. To inactivate each trypsin, 3 μL aprotinin with a concentration of 2.3 mg/mL was

149

directly added to each sample taken during hydrolysis, resulting in a molar ratio of trypsin to

× 100 %



150

aprotinin of 1:1.5. The inactivation was confirmed for each trypsin using the pH-stat. For this,

151

150 μL aprotinin was added to a 10 mL protein solution (10 mg/mL) immediately after

152

trypsin was added. For each enzyme, the pH of the solution remained constant after the

153

addition of aprotinin, proving that all enzymes were successfully inhibited (data not shown).

154

Due to the dissolution of CO2 in protein solutions during hydrolysis, the consumption of

155

NaOH needs to be corrected. As a control experiment, 1 % [w powder/v] of α-LA and β-cas

156

solutions were incubated in a pH-stat in the absence of trypsin for 3 h. The added volume of

157

NaOH from each control experiment was subtracted from the added volume of NaOH at all

158

time points during hydrolysis. Both 1st and 2nd order reaction equations were used to fit the

159

experimental DHstat curves. Using 2nd order reaction equation, the R2 of the fit against time

160

(R2=1-SSresidual/SStotal, SS means the sum of squared errors) was higher than using 1st order

161

reaction equation. Thus, the 2nd order reaction equation (equation 2) was used to fit the

162

experimental data,

163

(2) [%] = , − , /(1 +

164

where the maximum degree of hydrolysis (DHmax,fit) and the overall hydrolysis rate constant

165

(k)*+, '( [1/s]=khydr·DHmax,fit) were fitting parameters. All hydrolyses were performed in duplicate,

166

and samples were taken at the same DH points during each individual experiment. All

167

hydrolysates were analysed by adapting the method as developed previously by Butré et.al 3.

168

Estimation of the number of efficiently hydrolysed cleavage site

!"#

× , × $)

169

Based on the fitting parameter DHmax,fit (derived from equation 2), the percentage of CSs

170

that were efficiently hydrolysed by the enzyme, or in other words the percentage of high

171

selectivity sites (HSSs) of the enzyme, was estimated from equation (3) 1.

172

012!0 (3) --.#/01 (%) =

:;:9;

34567,89

:;:9;

34567,?:

× 100 %


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173

The superscripts and subscript of the parameters refer to the enzyme-substrate

174

combinations.

175

Reversed phase ultra-high performance liquid chromatography (RP-UPLC)

176

The hydrolysates were analysed on an H class Acquity UPLC® system (Waters, Milford,

177

MA, USA) equipped with a BEH C18 column (1.7 μm, 2.1×100 mm, Waters), connected to an

178

Acquity UPLC® PDA detector (Waters). The disulphide bridges were reduced by incubating

179

the samples at a protein concentration of 0.5 % for 2 h with 100 mM ditiothreitol (DTT) in 50

180

mM Tris-HCl buffer at pH 8.0. The reduced samples were further diluted to a protein

181

concentration of 0.1 % [w/v] and centrifuged (10 min, 14,000×g, 20 °C) before injection (4

182

μL).

183

2.2.4 Electron spray ionization time of flight mass spectrometry (ESI-Q-TOF-MS)

184

The MS and MS/MS (MSe) data of the peptides were collected with an online SYNAPT

185

G2-Si high definition mass spectrometry (Waters) coupled to the RP-UPLC system. The MS

186

system was calibrated with sodium iodide. Online lock mass data (angiotensin II, [M+2H]2+:

187

523.7751) was acquired 2. Based on the differences between measured and theoretical lock

188

mass, corrections were applied on the m/z of peptides during data collection. The data were

189

analysed manually using MassLynx software v4.1 (Waters) and UNIFI software v1.8 (Waters).

190

Estimation of the number of possible peptides

191

For a protein with N cleavage sites, the theoretical number of possible peptides

192

(NPPmax,theo) was calculated based on enzyme specificity using equation 4 13,

193

(4) @@,0/ = ( + 1)( + 2)/2

194

where N is the number of CSs in the protein sequence. Experimentally, the enzyme only

195

efficiently hydrolysed a few -high selectivity- CSs (HSSs). Therefore, a corrected number of



Page 10 of 52

196

possible peptides (NPPmax,HSS) based on enzyme selectivity was calculated by replacing N in

197

the equation 4 by the number of HSS (#HSS) estimated using equation 3.

198

Peptide identification and quantification

199

The mass tolerance between the theoretical mass and the measured mass for the

200

accepted annotation was set at 100 ppm. Two AA modifications, i.e. methionine oxidation

201

(+16 Da) in α-LA and the phosphorylation (+80 Da per phosphoserine) in β-cas, were taken

202

into account. The peptides were annotated based on the MS spectra and were confirmed by

203

identifying the b and y fragments in the MS/MS spectra when possible. In the case of co-

204

elution, the concentration of each of the co-eluting peptides was calculated by using the

205

intensity of total ion count to divide the UV area at 214 nm. The concentration of each

206

peptide was calculated using equation 5,

207

(5) B.0."0 [CD] = K

208

where Cpeptide [μM] is the peptide concentration, A214 [μAU·min] is the UV area at 214 nm,

209

Vinj [μL] is the injected volume of the sample, Q [μL·min-1] is the flow rate and l [cm] is the

210

path length of the UV cell, which is 1 cm according to the manufacturer. ε214 [1/M/cm] is the

211

molar extinction coefficient at 214 nm, which was calculated as described previously 14. Due

212

to the multiple reflection by the coating of the UV cell, the effective path length of the light

213

through the cell is not the same as the length specified by the producer. This issue was also

214

reported in earlier studies 15-17. To correct for this effect, the cell constant of the UV detector

215

(kcell), was determined using a series of standard solutions made by β-lactoglobulin, β-cas

216

and angiotensin II, and the approach as described elsewhere

217

measured and expected A214 was taken as the kcell value. For the UV cell used in this study,

218

the kcell was reported to be 0.78 2. The linear region of the peak area (A214) in the UV

219

detector ranges from 5×101 to 6×105 μAU·min 2. Therefore, peptide quantification was

EFGH ∙J ∙L∙M FGH 9;N ∙OP:QQ


18

. The ratio between the

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220

performed for peptides with A214≥5×101 μAU·min. In all hydrolysates, the total peak area in

221

the chromatograms was 95±6 % of the expected value, and of the total A214, 97±3 % was

222

assigned to annotated peptides. The standard error on the peptide concentration for one

223

hydrolysate injected in triplicate was reported to be 6 % using the same quantification

224

method as described above 3. The selectivity determined from duplicate hydrolyses was

225

reported to have a standard error of 15 % 3. In this work, of the duplicate experiments, the

226

average standard error of the peptide concentration was ~15 %.

227

Enzyme affinity towards intact proteins

228

The percentage of remaining intact protein in the hydrolysates was plotted against

229

hydrolysis time. The initial hydrolysis rate constants of intact protein (k WSX*YW,Z,[TWRS [1/s]) were RSTUVT

230

calculated from the initial slope of the curve (the linear slope of the first 3 points as a

231

function of time). According to the Linderstrøm-Lang theory, the amount of intact protein as

232

a function of DH illustrates the enzyme affinity towards intact proteins relative to the

233

enzyme affinity towards intermediate peptides

234

intact protein in the hydrolysates was also plotted against DHstat [%]. Because the hydrolyses

235

by the three trypsins did not reach the same DHmax,fit, the DHstat was normalised by dividing

236

by DHmax,fit.

237

Quality check of the annotation and quantification of the peptide analysis

19

. Based on this theory, the percentage of

238

The quality of peptide analysis was analysed using three parameters, i.e. amino acid,

239

peptide and molar sequence coverages, which were calculated by equations 6, 7 and 8,

240

respectively 3. These checks were performed for all hydrolysates. For each enzyme-substrate

241

combination, the average value for each parameter was calculated as the average of that

242

parameter for the duplicate experiments at all DH values.

243

(6) \]^_` ab^c defge_be b`heiaje [%] =

#l1ml0 11/0" EE #EE>:9;

× 100 %



244

(7) @en$^ce defge_be b`heiaje [%] = #EE(11/0" .0."0)o#EE(1p .0."0) × 100 % #EE(11/0" .0."0)

245

∑(x; yxz )F u v {#||>:9; yG}s s (8) D`qai defge_be b`heiaje [%] = 1 − × 100 % ~z t s s r

246

Cn [μM] represents the concentration of each individual AA (n) in the protein sequence; C0

247

[μM] is the initial injected protein concentration and #AAprotein is the number of AA in the

248

sequence of the parental protein 3. The injected initial concentrations (C0) were 59 µM and

249

34 μM for α-LA and β-cas, respectively (calculated based on the protein content and purity

250

listed in Table 2). In the samples at DH 0 % of α-LA and β-cas, the experimental C0 were

251

measured to be 58±2 and 33±1 μM, respectively, using equation 5. The missing peptides are

252

the peptides that were not found but should be present given the annotated peptides 3. To

253

further check the quality of peptide analysis, for each hydrolysate, the degree of hydrolysis

254

based on peptide analysis (DHMS) was calculated using equation 9,

255

∑~

9, (9) [%] = ×#.0."0 /1"×~ × 100 % z

256

where #peptide bonds stands for the number of peptide bonds and the Ci,t is the

257

concentration of the cleavage site products, calculated using equation 10.

258

Determination of the hydrolysis rate constants

259

For each CS, the concentration of cleavage site products (Ci,t) that originate from the

260

hydrolysis of that peptide bond was calculated using equation 10.

261

(10) B, [CD] = ∑B.0."0 [ − ] ^ = − 1 ∪ ^ =

262

where Ci,t [μM] refers to the concentration of CS products formed at each time point t. i

263

equals the sum of the concentrations of all peptides of sequence [x-y], for which i=(x-1) or

264

i=y. The apparent hydrolysis rate constant was obtained by fitting the equation of 2nd order

265

reaction equation (equation 11) to the experimental data 2, 12 ACS Paragon Plus Environment

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(11) B,012!0,.#/01 [CD] = 2 ∙ B012!0,.#/01 − B012!0,.#/01 /(1 +

012!0,.#/01 ,..

× B

012!0,.#/01

× $)

267

where both CWSX*YW,Z,[TWRS [μM] and kWSX*YW,Z,[TWRS [1/s/μMprotein] are fitting parameters, and the R,UZZ

268

superscripts refer to the enzyme-substrate combination. The apparent hydrolysis rate

269

constant

270

constant kWSX*YW,Z,[TWRS [1/s/mgprotein] of each hydrolysis was calculated using equation 12, R,V

271

(12)

272

where mE [mg] is the mass of the enzyme added for the hydrolysis. The Ci,t was also

273

calculated based on the concentration of remaining non-hydrolysed CSs (Cremain,t) (Ci,t=C0-

274

Cremain,t) in the hydrolysates. Hydrolysis rate constants calculated based on the two methods

275

were compared.

276

Enzyme selectivity

k R,UZZ

WSX*YW,Z,[TWRS

012!0,.#/01 ,

=

× C

WSX*YW,Z,[TWRS

is referred to as

k R,UZZ,V

WSX*YW,Z,[TWRS

[s-1]. The hydrolysis rate

:;:9;

O9,6,P

277

The selectivity [%] of enzymes towards CSs present in the two substrate proteins was

278

calculated by dividing the hydrolysis rate constant kWSX*YW,Z,[TWRS of each CS by the sum of R,V

279

hydrolysis rate constants for all CSs for that enzyme-substrate combination (equation 13) 2.

280

(13) -eqeb$^h^$ 012!0,.#/01 [%] =

281

In a recent work for bovine trypsin, the high (HSSs), intermediate (ISSs) and low/zero (LSSs)

282

selectivity sites in α-LA and β-cas were clustered using the Matlab method k-means

283

clustering 2. As a partitioning method, the k-means clustering uses the squared Euclidean

284

metric to determine the distances and the k-means++ algorithm for the cluster centre

285

initialization. It is used to find clusters that divide data points with minimized total sum of

286

distances. To compare hydrolysis rate constants of porcine and human trypsins towards CSs

287

with those of bovine trypsin, for each substrate, the lowest hydrolysis rate constant of

288

bovine trypsin towards CSs in each category (HSS, ISS and LSS) was set as the thresholds.

:;:9;

O9

:;:9;

∑(O9

:;:9;

×~z

:;:9;

×~z


)

× 100 %


289

Using the thresholds, the number of HSSs, ISSs and LSSs of porcine and human trypsins

290

towards CSs in α-LA and β-cas were determined.

291

Prediction of maximum degree of hydrolysis

292

of ISS and LSS were Previously, it was found that for bovine trypsin, the average k,Z,[TWRS R,V

293

~24 % and ~3 % of the average k ,Z,[TWRS of HSSs, respectively 2. It means that within the time R,V

294

of the hydrolysis experiment when most HSS were hydrolysed, ~24 % and ~3 % of the ISSs

295

and LSSs were hydrolysed, respectively. Hence, the predicted maximum degree of hydrolysis

296

(DHmax,pre) based on enzyme selectivity can be calculated using equation 14 2.

297

(14) ,.#0 [%] =

298

where #HSS, #ISS and #LSS stand for the number of high, intermediate and low/zero

299

selectivity sites, respectively. Since the #HSS, #ISS and #LSS of porcine and human tryptic

300

hydrolysis of α-LA and β-cas were obtained based on the thresholds set from bovine trypsin

301

selectivity, their DHmax,pre were also predicted using equation 14.

302

Relation between binding site sequence and enzyme selectivity towards CSs

#4o.×#o.×# #.0."0 /1"

× 100 %

303

The correlation between the binding site sequence and the hydrolysis rate constant was

304

analysed using the information for all CSs. For this, the standard errors of the molecular

305

weight, hydrophobic moment and charge of the AA at each binding site position were

306

analysed, using the same approach as reported previously 2. The scale of hydrophobic

307

moments ranged from most hydrophilic AA (arginine, -1.8), to the most hydrophobic AA

308

(isoleucine, 0.73)

309

which were the CSs, and glutamic and aspartic acids were negatively charged. Histidine was

310

not included as a charged AA because only 0.9 % of histidine was charged at pH 8. In

311

addition, cysteine was considered as a charged AA, because the sulfhydryl group was

312

charged at pH 8. The sulfhydryl group of cysteine could be free in the intact proteins, or

20

. For the analysis at pH 8, arginine and lysine were positively charged,


Page 14 of 52

Page 15 of 52


21

313

could be released as a result of disulphide bridge shuffling

. If the sulfhydryl group of

314

cysteine was involved in a disulphide bridge, it would not carry a charge, but it may still

315

affect the hydrolysis due to steric hindrance for enzyme-substrate binding.

316

Results and discussion

317

General description of α-LA and β-cas hydrolyses by the three trypsins

318

The hydrolysis curves (DHstat versus time) of α-LA by bovine, porcine and human trypsins

319

were fitted using equation 2. The overall hydrolysis rate constants (k //(, ) of hydrolysis '(

320

of α-LA by bovine (1.6(±0.2)·10-3/s)

321

higher than by human trypsin (1.1(±0.3)·10-3/s). Larger differences were found in the

322

DHmax,exp of hydrolysis by the three trypsins. The DHmax,fit values derived from equation 2 of

323

hydrolysis by the three trypsins were compared. For porcine trypsin, the DHmax,fit of α-LA

324

hydrolysis (10.0±0.3 %) (Figure 1A) was similar to the DHmax,theo (10.7 %) (Table 2). This

325

suggested that porcine trypsin efficiently cleaved ~94 % of the CSs in α-LA, suggesting that

326

around 12 out of 13 CSs were high selectivity sites (HSSs, equation 3). The DHmax,fit of α-LA

327

hydrolysed by human or bovine trypsins were 6.8±0.1 % and 5.1±0.2 % 2, respectively, which

328

were much lower than the DHmax,theo. Based on the DHmax,fit, for bovine or human tryptic

329

hydrolysis, only 48 % (6 out of 13) and 63 % (8 out of 13) of the CSs in α-LA, respectively,

330

were estimated to be HSSs. That porcine tryptic hydrolysis reached DHmax,theo but bovine

331

tryptic hydrolysis did not was in line with the data reported in literature. The DHmax,exp of

332

porcine tryptic hydrolysis of whey protein concentrate at 50 °C with a pH of 8.0 was reported

333

to be 14 %, which was slightly higher than the DHmax,theo (11.2 %) 22. The hydrolysis of whey

334

protein isolate by bovine trypsin performed at 37 °C and pH 7.8 reached a DHmax,exp of 7 %,

335

corresponding to ~63 % of the DHmax,exp 23.

2

or porcine (1.7(±0.0)·10-3/s) trypsins were slightly



336

) of βSimilarly as for α-LA hydrolysis, the overall hydrolysis rate constants (k //(,VU '(

337

cas by bovine (9.6(±0.4)·10-3/s)

2

or porcine (7.1(±0.7)·10-3/s) trypsins were slightly higher

338

than by human trypsin (6.4(±0.2)·10-3/s). For all enzymes, the hydrolysis rate constants of β-

339

cas hydrolysis were ~5 times higher than those of α-LA hydrolysis. The DHmax,fit of β-cas by

340

bovine, porcine or human trypsins were 4.3±0.1 % 2, 6.7±0.3 % and 5.3±0.1 %, respectively

341

(Figure 1B). When the β-cas hydrolysis by bovine trypsin was performed at 30 °C with a pH

342

of 7.6, the DHmax,exp was reported to be 5 % 24. This value was similar to the value obtained in

343

this study. Similarly as for α-LA hydrolysis, there was a difference in the number of estimated

344

HSSs for bovine, porcine or human trypsins, which were 60 % (9 out of 15), 93 % (14 out of

345

15) and 74 % (11 out of 15) of the CSs in β-cas, respectively. For α-LA hydrolysis, similar

346

initial hydrolysis rate constants were found for bovine, porcine and human trypsins

347

(k//(, ): 0.14, 0.18 and 0.10/s, respectively (Figure 2A). The percentage of remaining RSTUVT

348

intact protein in the hydrolysates as a function of DHstat/DHmax,fit indicated that the affinities

349

of three enzymes towards intact α-LA were also similar (Figure 2B). The k //(,VU and RSTUVT

350

enzyme affinities towards the intact β-cas for the three enzymes were also similar (Figures

351

2C and 2D). Between hydrolyses of the two proteins, some differences were observed.

352

Firstly, the k //(,VU of intact β-cas hydrolysed by bovine, porcine or human trypsins were RSTUVT

353

1.2, 2.5, 1.5/s, respectively, which were 8-14 times higher than the k //(, of intact α-LA. RSTUVT

354

Secondly, at DHstat 1 %, all intact β-cas molecules were hydrolysed by the three trypsins

355

(Figure 2D). Since a DHstat of 1 % means on average that ~2 peptide bonds were hydrolysed

356

in 208 peptide bonds (in β-cas), this suggests that there were 1 or 2 peptide bond(s) that

357

was/were much more susceptible to hydrolysis in intact β-cas than other peptide bonds. This

358

was not observed in α-LA hydrolysis by any of the three trypsins. Because apo α-lactalbumin

359

denatures at 37 °C (the hydrolysis temperature) and β-casein is a random coil protein, the 16 ACS Paragon Plus Environment

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360

difference in hydrolysis between the two proteins was not caused by the compactness of the

361

protein tertiary structure. It is possible that β-casein molecules formed micellar structure in

362

solution, which may have caused the differences between the two proteins in overall

363

hydrolysis rates and hydrolysis rates of intact proteins.

364

Since no difference in enzyme affinity towards intact proteins was found in hydrolysis by

365

the three trypsins, the difference in DHmax,exp was most likely due to variations in the

366

amounts of HSSs of each hydrolysis. This was analysed in detail using a quantitative peptide

367

analysis.

368

Peptide analysis of α-LA hydrolysis by the three trypsins

369

Peptide annotation of α-LA hydrolysis by the three trypsins

370

Based on the estimated number of HSSs, the predicted maximum number of peptides

371

(NPPmax,HSS) was 28, 91 and 45 for bovine, porcine and human tryptic hydrolysates,

372

respectively (equation 4). Experimentally, 23 2, 30 and 31 unique peptides were annotated in

373

bovine, porcine and human tryptic hydrolysates of α-LA, respectively (Table 2 and Appendix

374

A). The numbers of annotated peptides were lower than the NPPmax,HSS, particularly for

375

porcine trypsin. This could be because a large number of intermediate peptides that should

376

be formed during the hydrolysis only accumulated to very low concentrations (only 5 % of

377

the expected A214 was not annotated), e.g. below detection or quantification limit. The

378

quality of peptide annotation and quantification was analysed to identify how much of the

379

total hydrolysate was represented by the quantified peptides.

380

Quality check of the peptide analysis of α-LA by the three trypsins

381

The quality of peptide analysis was considered to be sufficient, based on the analysis by

382

four parameters: the average AA, peptide and molar sequence coverages, and the DH

383

calculated from the peptide analysis (DHMS). The AA sequence coverages of all samples were 17 ACS Paragon Plus Environment


Page 18 of 52

384

100 % (Table 3). The average peptide sequence coverages for bovine, porcine and human

385

tryptic hydrolysates were comparable: 96±3 % 2, 90±2 % and 95±5 %, respectively. The

386

average molar sequence coverages for bovine, porcine and human tryptic hydrolysates were

387

80±9 % 2, 70±11 % and 72±7 %, respectively (Table 3 and Appendix C). The sequence

388

coverages in this work were higher than the thresholds set based on the data obtained from

389

previous studies

390

coverages, respectively 2. A further confirmation was obtained by comparing the DHMS with

391

the DH values taken from the pH-stat titration (DHstat). Averaged over all hydrolysates, the

392

standard error between DHstat and DHMS was ~7.3 % (Figure 3A). For the 3 porcine tryptic

393

hydrolysates towards the end of the hydrolysis, the DHMS were smaller than the DHstat, which

394

was also reflected by the low molar sequence coverages (≤60 %) (Appendix C). This only had

395

a minor effect on the fitting parameters, because for the fitting of data with the equation of

396

2nd order reaction, the results were mostly determined by the initial 5 data points. Those 5

397

hydrolysates had high molar sequence coverages (≥70%).

3, 25-26

, i.e. 80% and 70 % for the average peptide and molar sequence

398

In bovine tryptic hydrolysates, only specific cleavages were found 2, while in porcine and

399

human tryptic hydrolysates, 3 and 1 a-specific cleavage(s) were identified, respectively. Such

400

a-specific cleavages were previously found in the later stage of whey protein isolate

401

hydrolysis by Bacillus licheniformis protease (BLP)

402

hydrolysed only in certain specific peptides. Later on, these peptides were synthesized and

403

incubated in the absence and presence of BLP 27. Even without the presence of enzyme, the

404

a-specific cleavages still occurred on those unstable peptide bonds

405

degradation of intrinsically unstable peptides bonds were referred to as the “spontaneous

406

cleavage”

27

27

. These sites were observed to be

27

. This autolytic

. In this study, the peptides that formed after a-specific cleavages were found


Page 19 of 52


407

only at the end of hydrolysis, and these sites were cleaved to low extents (≤6 µM). Therefore,

408

they were not included as CSs.

409

Peptide release kinetics of α-LA hydrolysis by the three trypsins

410

In all hydrolyses, the peptide concentrations were followed during hydrolysis (Appendix

411

E). The differences between the hydrolyses by the three enzymes were illustrated for the

412

peptides α-LA[14-16] and α-LA[1-10] (Figures 4A and 4B). During bovine and porcine tryptic

413

hydrolysis, α-LA[14-16] was released rapidly. Because α-LA[14-16] was a final peptide, i.e. a

414

peptide that did not contain CSs, the concentration only increased. In porcine tryptic

415

hydrolysis, the concentration of α-LA[14-16] in the final hydrolysate reached the injected

416

concentration of α-LA (C0=59 µM) (Figure 4A). This illustrated that CSs K13-14 and K16-17

417

were efficiently hydrolysed by porcine trypsin. During bovine tryptic hydrolysis, the

418

concentration of peptide α-LA[14-16] reached ~42 µM at the end of hydrolysis. During

419

human tryptic hydrolysis, peptide α-LA[14-16] was released slowly and the concentration

420

reached ~34 µM at the end. Peptide α-LA[1-10] is an intermediate peptide, of which the

421

concentration can decrease after the hydrolysis of CS K5-6 (Figure 4B). For hydrolysis by all

422

trypsins, α-LA[1-10] formed rapidly in the beginning. However, for the hydrolysis by different

423

trypsins, the concentration reached different values before it started to decrease. In final

424

porcine and human tryptic hydrolysates, α-LA[1-10] was not found, which means that K5-6

425

was fully hydrolysed. Unlike hydrolysis by these two trypsins, the concentration of α-LA[1-10]

426

in the final bovine tryptic hydrolysate was ~10 µM. This means that K5-6 was not fully

427

hydrolysed by bovine trypsin. This was also seen by the final cleavage site products of K5-6,

428

which will be discussed in the next section.

429

Hydrolysis rate constants of CS in α-LA by the three trypsins



430

Based on the peptide concentrations at each DH, the concentration of cleavage site

431

products of each CS during α-LA hydrolysis was determined. There were clear differences in

432

the hydrolysis rate of each trypsin of individual CSs, as shown for instance for CS K13-14

433

(Figure 4C). This CS was rapidly hydrolysed by bovine and porcine trypsins, but not by human

434

trypsin. CS K13-14 was fully hydrolysed by porcine trypsin as indicated by the final CS

435

products concentration, as well as by the peptide release kinetics of α-LA[14-16]. For CS K5-6,

436

the concentration of the CS products increased rapidly for hydrolysis by all trypsins (Figure

437

4D), which was expected based on the peptide release kinetics of peptide α-LA[1-10]

438

discussed above. Still, while for porcine and human tryptic hydrolyses, the concentrations of

439

CS products at the end of hydrolysis reached 59 and 55 µM, respectively, for bovine tryptic

440

hydrolysis, it only reached 34 µM. During porcine tryptic hydrolyses, most CSs reached C0,

441

meaning that they were fully hydrolysed. This was not the case for bovine and human

442

trypsins. For some CSs, the hydrolysis rate constants of bovine and human trypsins were low,

443

meaning that they were not hydrolysed yet within the experiment time. In other cases, the

444

molar sequence coverages of some part of the sequence were not complete, resulting in a

445

low concentration of CS products. In addition, for bovine and human tryptic hydrolysis, there

446

was ~12 % intact α-LA not hydrolysed at the end of hydrolysis. This means that the

447

remaining non-hydrolysed CSs were in the remaining intact α-LA.

448

From the CS products formation kinetics, hydrolysis rate constants (k//(, ) of the R,V

449

three enzymes towards CSs in α-LA were determined (Table 4). Differences between the

450

enzymes were seen for instance by the hydrolysis kinetics of CS K62-63. This CS was

451

efficiently hydrolysed by porcine trypsin (1.7·10-3/s/mgenzyme). However, it was not

452

hydrolysed by bovine trypsin at all, and was only slowly hydrolysed by human trypsin

453

(0.07·10-3/s/mgenzyme). Overall, the total hydrolysis rate constants of porcine trypsin for CSs 20 ACS Paragon Plus Environment

Page 20 of 52

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454

) were ~2 times higher than those of bovine and human trypsins. The k , in α-LA (k, R,V R,V

455

for bovine trypsin towards CSs in α-LA were equally distributed among the range of 10-5, 10-4

456

and 10-3 /s/mgenzyme. The similar range of k, was found for k (, of human trypsin. R,V R,V

457

Unlike these two trypsins, for porcine trypsin, 10 out of 13 CS had similar hydrolysis rate

458

constants, which were all ≥10-3/s/mgenzyme. For porcine and human trypsins, the hydrolysis

459

rate constants of CSs in α-LA were also calculated based on the concentration of remaining

460

non-hydrolysed CS (Cremain,t) in the hydrolysates. For CSs in the part of AA sequence where

461

the molecular sequence coverages were high, the hydrolysis rate constants calculated based

462

on the two methods have an average standard error of 26 %. This was 10 % higher than the

463

average standard error of hydrolysates in duplicate experiments (16 %). The hydrolysis rate

464

constants of CSs calculated using both methods were used to further calculate the enzyme

465

selectivity.

466

Using the same approach as the peptide analysis of α-LA hydrolysates, the hydrolysis

467

kinetics, peptide release kinetics and hydrolysis rate constants of the three trypsins towards

468

CSs in β-cas were analysed.

469

Peptide analysis of β-cas hydrolysis by the three trypsins

470

Peptide annotation of the hydrolysis of β-cas by the three trypsins

471

Based on the estimated number of HSSs, the predicted maximum number of peptides

472

(NPPmax,pre) was 55, 120 and 78 for bovine, porcine and human tryptic hydrolysates of β-cas,

473

respectively. Experimentally, 34 2, 35 and 40 unique peptides were annotated in bovine,

474

porcine and human tryptic hydrolysates, respectively (Table 2 and Appendix B). Similarly as

475

for α-LA hydrolysis, the annotated numbers of peptides were lower than prediction.

476

Quality check of the peptide analysis of β-cas by the three trypsins



477

Overall, for all β-cas hydrolysates, the average AA, peptide and molar sequence

478

coverages were in the same range as those for α-LA (Table 3 and Appendix D). Only porcine

479

tryptic hydrolysates at DH 6.7 % had an AA sequence coverage less than 100 %. This was

480

because this hydrolysate mainly contained final peptides, where dipeptides β-cas[98-99] and

481

β-cas[106-107], were not found in the chromatograms. Because dipeptides have low molar

482

extinction coefficients, the loss of UV signal was not visible from the total UV signal. The

483

quality of peptide quantification was further confirmed by the agreement between the

484

calculated DHMS of each β-cas hydrolysate and DHstat (Figure 3B). Averaged over all samples,

485

the standard error between the DHstat and DHMS was ~7.7 %. In bovine, porcine and human

486

hydrolysates of β-cas, peptides with 4, 5 and 4 a-specific cleavages were found, respectively.

487

Due to low concentrations of CS products for these sites (≤8 µM), they were not included as

488

CS.

489

Peptide annotation of the hydrolysis of β-cas by the three trypsins

490

The differences in peptide release kinetics during hydrolysis of β-cas by the three

491

trypsins were compared (Appendix F). This was illustrated for the peptides β-cas[108-113]

492

and β-cas[183-209] (Figures 5A and 5B). As expected, for porcine tryptic hydrolysis, the final

493

peptide β-cas[108-113] formed rapidly in the beginning and reached ~35 μM after 1000 s,

494

which was close to the initial injected concentration (34 μM) (Figure 5A). This indicated that

495

CSs K108-109 and K114-115 were fully hydrolysed by porcine trypsin. During bovine tryptic

496

hydrolysis, β-cas[108-113] was released gradually and the concentration reached ~23 μM.

497

For hydrolysis by human trypsin, β-cas[108-113] was not formed in the first ~3500 s, but was

498

so rapidly accumulated after this point that it still reached the initial injected concentration

499

(34 μM) before the end of the experiment. Previously, such delay in the release kinetics of

500

peptides was attributed to the “demasking” phenomonon. Demasking describes that during 22 ACS Paragon Plus Environment

Page 22 of 52

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501

protein enzymatic hydrolysis some CSs may only become accessible after cleavage of other

502

peptide bonds in the protein

503

209], increased rapidly from t=0 and started to decrease at DH 2 % in porcine tryptic

504

hydrolysis, and was completely degraded at ~1000 s (Figure 5B). For both bovine and human

505

tryptic hydrolyses, β-cas[183-209] formed gradually during the hydrolysis and started to

506

decrease at ~400 and ~800 s, respectively. There was ~5 µM of β-cas[183-209] remained in

507

the final bovine tryptic hydrolysate, whereas for the other two trypsins, it was completely

508

degraded.

509

Hydrolysis rate constants of CSs in β-cas by the three trypsins

28

. The concentration of the intermediate peptide β-cas[183-

510

During porcine tryptic hydrolysis, the concentration of CS products of K113-114

511

increased rapidly at the start, reaching the initial injected concentration (34 µM) within

512

~1500 s (Figure 5C). The hydrolysis of K113-114 by bovine trypsin was slower than by

513

porcine trypsin. By bovine trypsin, the concentration of the CS products only reached ~27

514

μM at the end. For human trypsin, as indicated by the peptide release kinetics of β-cas[108-

515

113], the cleavage of K113-114 was masked in intact β-cas. Indeed, no CS products of K113-

516

114 were formed in the beginning of hydrolysis. However, after DH 4 %, the concentration

517

increased rapidly and reached ~33 μM (C0=34 µM). This indicated that human trypsin could

518

hydrolyse K113-114 once it was demasked. This also explained why the fit using the 2nd

519

order reaction equation was not good for this CS. Clear evidence of demasking effects were

520

observed in only 4 cases (R202-203 for the three enzymes and K113-114 for human trypsin)

521

in all hydrolyses. The equation for the consecutive reaction, as describe previously

522

used to calculate the hydrolysis rate constants of the CSs. For CS K183-184, all trypsins

523

hydrolysed the CS efficiently, illustrated by the fact that all the CS products in the end of


29

, was


524

hydrolysis reached C0 (Figures 5D). However, porcine trypsin hydrolysed the K183-184 faster

525

than the other two trypsins.

526

The hydrolysis rate constants (k //(,VU ) of all CSs in β-cas by the three enzymes were R,V

527

determined (Table 5). Similarly as for α-LA hydrolysis, the total hydrolysis rate constants

528

(k,VU ) of porcine trypsin towards CSs in β-cas were higher than bovine and human R,V

529

trypsins. In the final hydrolysate of β-cas during hydrolysis by porcine trypsin, mainly final

530

peptides were found, indicating that almost all CSs were fully hydrolysed. This means that

531

,VU the differences in k,VU were not from the differences in C but almost entirely from R,V

532

the differences in k,VU . None of the three enzymes hydrolysed the CS R1-2. For the R,UZZ

533

hydrolysed CSs, bovine and human trypsins were both more selective than porcine trypsin,

534

but the preferred CSs were not the same. As shown in Figure 2B, in all hydrolyses, there

535

were 1 or 2 CSs highly preferred by each trypsin, resulting in almost complete hydrolysis of

536

intact β-cas at DHstat 1 %. This CS highly preferred by bovine trypsin was reported to be K169-

537

170 2. In this work, K169-170 was also found to be highly preferred by porcine trypsin,

538

indicated by the fact that at DHstat 1 % the CS products of K169-170 reached C0. Unlike

539

bovine and porcine trypsins, the highly preferred CS by human trypsin was R25-26. For

540

porcine and human trypsins, the hydrolysis rate constants of CSs were also calculated based

541

on the concentration of remaining non-hydrolysed CSs in the hydrolysates. For CSs that were

542

in the part of the AA sequence with high molar sequence coverages, the hydrolysis rate

543

constants of the CSs determined based on both methods had an average standard error of

544

28 %.

545

It should be noted that even for the HSSs, the hydrolysis rate constants of the three

546

trypsins towards CSs in β-cas were higher than those of CSs in α-LA. This might be due to the

547

differences in hydrolysis rate constants of intact proteins (kintact) during the hydrolysis. The 24 ACS Paragon Plus Environment

Page 24 of 52

Page 25 of 52


548

kintact values of the three trypsins of β-cas were 8-14 times higher than their kintact values of

549

α-LA. This was also reflected on the total hydrolysis rate constants of the two proteins. This

550

then suggests that the measured hydrolysis rate constant of CSs are the product of the kintact

551

and the hydrolysis rate constants of the CS in the peptides.

552

Enzyme selectivity and the prediction of maximum degree of hydrolysis

553

Based on the hydrolysis rate constants calculated by the concentrations of CS products

554

and the remaining non-hydrolysed CSs (Cremain,t) in the hydrolysates, the selectivities of

555

bovine, porcine and human trypsin towards CSs in α-LA and β-cas were calculated using

556

equation 13. Calculated based on the two methods, for the CSs in the part of AA sequence

557

where the molecular sequence coverages were high, the selectivity values had an average

558

standard error of 26 %. The selectivity values calculated based on the concentrations of CS

559

products were used for further comparison between the selectivity of each trypsin (Figures

560

6A and 6C). The selectivity values of porcine trypsin towards most CSs in α-LA were similar,

561

while the range of selectivity values for bovine and human trypsin selectivity was much

562

larger. This means that bovine and human trypsins were more selective than porcine trypsin

563

towards CSs in α-LA (Figure 6A). For β-cas hydrolysis, the total hydrolysis rate constant of

564

porcine tryptic hydrolysis was 3 times higher than the bovine and human tryptic hydrolysis

565

(Figure 6D). Although relatively the ranges of selectivity values were similar for hydrolysis by

566

the three trypsins, porcine trypsin efficiently hydrolysed more CSs than the other two

567

trypsins. This finding was in line with another study, which reported a human serum albumin

568

hydrolysate by bovine trypsin contained ~60 % more peptides with missed CSs in the AA

569

sequence than by porcine trypsin 6.

570

Based on the threshold k//(, values from the results of bovine trypsin selectivity, R,V

571

the high (HSSs), intermediate (ISSs) and low/zero (LSSs) selectivity sites of porcine and 25 ACS Paragon Plus Environment


572

human trypsins towards CSs in α-LA and β-cas were categorised (Table 6). For CSs in α-LA,

573

the amount of HSSs for each trypsin was close to but slightly lower than the prediction based

574

on DHmax,fit, which was 6, 12 and 8 for bovine, porcine and human trypsins, respectively. This

575

was also observed for β-cas hydrolysis. That lower number of HSSs obtained experimentally

576

was due to the fact that ISSs also contributed to the DHmax,fit. Based on the numbers of HSSs,

577

ISSs and LSSs, the predicted maximum degree of hydrolysis (DHmax,pre) based on enzyme

578

selectivity was calculated using equation 14, and compared with DHmax,exp (Figure 7). The

579

average standard error (|DHmax,pre/theo-DHmax,exp|/DHmax,exp×100 %) between the predicted

580

DHmax,pre based on selectivity and the experimental DHmax,exp was ~14 %, which was ~4 times

581

more accurate than the prediction based on enzyme specificity (DHmax,theo).

582

Based on coherent outcomes of α-LA and β-cas hydrolysis by bovine, porcine or human

583

trypsins, it is concluded that the differences in DHmax,exp of hydrolysis by the three trypsins

584

can be explained by their differences in enzyme selectivity. Based on the percentage of HSSs

585

of the total CSs, for the two substrates, bovine, porcine and human trypsin efficiently

586

hydrolysed 43 %, 75 % and 50 %, respectively.

587

Link between binding site sequence and selectivity of porcine and human trypsins

588

Using the rules of bovine trypsin selectivity, 57 % (16 out of 28) and 50 % (14 out of 28)

589

of CSs for porcine and human trypsin selectivity, respectively, were correctly predicted

590

(Table 1). To improve the prediction, the Link between the binding site sequence and the

591

selectivity of porcine and human trypsins was studied. The selectivity of porcine and human

592

trypsin towards CSs, was not related to the hydrophobicity and molecular weight of the AAs

593

on the binding site sequence. For both trypsins, the standard errors of the molecular weights

594

of AAs in all positions in the binding site sequence in the 3 categories (HSS, ISS and LSS) were

595

similar (10-22 %), and close to the standard error over all 20 protein AAs (22 %). For 26 ACS Paragon Plus Environment

Page 26 of 52

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596

hydrophobicity, the absolute values of standard errors for hydrophobic moments of all

597

binding site positions in the 3 categories were between 95 and 8220 %, and the absolute

598

standard error over all 20 protein AA is ~420 %. There was a relation between selectivity of

599

porcine trypsin, and charge of the AAs on the P2 position. All 5 LSSs had a negative charged

600

AA on that position (Table 7). Out of the 21 HSSs, only 2 CSs (α-LA K62 and K98) had a

601

negative charged AA on P2 position (if the cysteine was taken as a charged AA). For all other

602

binding site positions, no clear differences were found between the HSSs and LSSs. This rule,

603

i.e. porcine trypsin efficiently hydrolyses CSs with no negatively charged AAs on P2 position

604

(Table 9), explained the porcine trypsin selectivity towards of 24 out of 28 (~86 %) CSs in α-

605

LA and β-cas. Positively charged AAs did not seem to hinder the porcine trypsin selectivity

606

since they were not present in the binding site sequences of the LSSs, but were mostly

607

present in the binding site sequences of the HSSs.

608

For human trypsin, it was found that 11 out of 15 ISSs and LSSs had a negative charged

609

AA on either P2 or P1’ positions of the binding site sequences (Table 8). For HSSs, only 3 out

610

of 13 CSs had negative charged AAs on either P2 or P1’ positions of the binding site

611

sequence (if cysteine was taken as a charged AA). This means that human trypsin selectivity

612

towards 21 out of 28 (~82 %) CS can be explained by the hindrance of negatively charged

613

AAs on either P2 or P1’ positions (Table 9). No clear observation was found on the effect of

614

positively charged AAs on the human trypsin selectivity. That porcine trypsin was only

615

hindered by a charged AA on 1 position of the binding site sequence explained why it

616

reached higher DHmax,exp than the other two trypsins.

617

In this article, for two proteins (α-lactalbumin and β-casein), large differences were

618

found in the DHmax,exp as well as the hydrolysate compositions between bovine, porcine and

619

human tryptic hydrolysis. Based on the full quantitative peptide analysis, porcine trypsin 27 ACS Paragon Plus Environment


620

efficiently hydrolysed ~78 % of the cleavage sites, while the values for bovine and human

621

trypsins were only ~47 % and ~53 %, respectively. These data explained why the porcine

622

tryptic hydrolysis reached ~90 % the theoretical maximum, while bovine and human tryptic

623

hydrolysis reached ~54 % and 68 %, respectively. This outcome means that the in vitro

624

protein digestibility determined by porcine tryptic hydrolysis should be ~2 times higher than

625

determined by bovine or by human tryptic hydrolysis. Knowing the enzyme selectivity, the

626

average standard error of the DHmax prediction decreased from 52 % (based solely on

627

enzyme specificity) to 13 %. Preliminary relations between the amino acid sequence of the

628

binding site positions and the selectivity of each trypsin were proposed, which would allow

629

estimations of the DHmax,exp of hydrolysis of other substrate proteins by these trypsins. This

630

work can help to explain the differences in the reported in vitro digestibility of proteins, and

631

to define expectations of the outcome of future experiments using these trypsins. It is a

632

step forward to obtain a better understanding of the hydrolysis process.


Page 28 of 52

Page 29 of 52


633

Abbreviation used

634

DH

Degree of hydrolysis

635

DHmax,theo

Theoretical maximum degree of hydrolysis

636

DHmax,exp

Experimental maximum degree of hydrolysis

637

DHmax,fit

Fitted maximum degree of hydrolysis

638

DHmax,pre

Predicted maximum degree of hydrolysis

639

kDH

Overall hydrolysis rate constant

640

kintact

Hydrolysis rate constant of intact proteins

641

ki,c

Hydrolysis rate constant of cleavage sites

642

C0

Initial injected protein concentration

643

HSS

High selectivity sites

644

ISS

Intermediate selectivity sites

645

LSS

Low/zero selectivity sites

646

CS

Cleavage site

647

AA

Amino acid

648

BT

Bovine trypsin

649

PT

Porcine trypsin

650

HT

Human trypsin

651

A214

UV peak area at 214 nm

652

RP-UPLC

Reversed phase ultra-high performance liquid chromatography

653

ESI-Q-TOF-MS

Electron spray ionization time of flight mass spectrometry

654

α-LA

α-Lactalbumin

655

β-cas

β-Casein

656 29 ACS Paragon Plus Environment


657

Acknowledgement

658

This work is supported by NanoNextNL, a micro and nanotechnology consortium of the

659

government of the Netherlands and 130 partners. This work is financially supported by

660

FrieslandCampina, The Netherlands.

661 662

Conflicts of interest: The authors declare that they have no conflict of interest.

663


Page 30 of 52

Page 31 of 52


664

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665

1.

666

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667

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670

3.

671

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672

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673

4.

674

of healthy volunteers, chronic alcoholics, and patients with pancreatitis and cancer of the

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pancreas. Gut 1979, 20 (10), 886-891.

676

5.

677

peptide release. Biochemical Engineering Journal 2013, 70, 88-96.

678

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Comprehensive analysis of protein digestion using six trypsins reveals the origin of trypsin as

680

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681

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682

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683

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684

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leading protease in proteomics. Mass Spectrometry Reviews 2013, 32 (6), 453-465.

686

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688

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porcine trypsins. Comparative Biochemistry and Physiology Part B: Biochemistry and

690

Molecular Biology 2002, 131 (3), 423-431.

691

11.

692

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693

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694

enzymatic hydrolysis of proteins. Process Biochemistry 2014, 49, 1903-1912.

Deng, Y.; Wierenga, P. A.; Schols, H. A.; Sforza, S.; Gruppen, H., Effect of Maillard

Deng, Y.; van der Veer, F.; Sforza, S.; Gruppen, H.; Wierenga, P. A., Towards

Butré, C. I.; Sforza, S.; Gruppen, H.; Wierenga, P. A., Introducing enzyme selectivity: A

Rinderknecht, H.; Renner, I. G.; Carmack, C., Trypsinogen variants in pancreatic juice

Fernández, A.; Riera, F., β-Lactoglobulin tryptic digestion: A model approach for

Walmsley, S. J.; Rudnick, P. A.; Liang, Y.; Dong, Q.; Stein, S. E.; Nesvizhskii, A. I.,

Keil, B., Specificity of proteolysis. Springer-Verlag Berlin Heidelberg, New York, USA:

Vandermarliere, E.; Mueller, M.; Martens, L., Getting intimate with trypsin, the

Schechter, I.; Berger, A., On the size of the active site in proteases. I. Papain.

Dallas Johnson, K.; Clark, A.; Marshall, S., A functional comparison of ovine and

Adler-Nissen, J., Enzymic hydrolysis of food proteins. Elsevier Applied Science

Butré, C. I.; Wierenga, P. A.; Gruppen, H., Influence of water availability on the



695

13.

Butré, C. I. Introducing enzyme selectivity as a quantitative parameter to describe the

696

effects of substrate concentration on protein hydrolysis. PhD thesis Wageningen University

697

& Research, Wageningen, the Netherlands, 2014.

698

14.

699

and peptides using UV absorption of the constituent amino acids at 214 nm to enable

700

quantitative reverse phase high-performance liquid chromatography-mass spectrometry

701

analysis. Journal of Agricultural and Food Chemistry 2007, 55 (14), 5445-5451.

702

15.

703

Optimization and evaluation of the performance of arrangements for UV detection in high-

704

resolution separations using fused-silica capillaries. Journal of Chromatography A 1991, 559

705

(1-2), 163-181.

706

16.

707

capillary electrophoresis. Journal of Chromatography A 1991, 543 (C), 439-449.

708

17.

709

for high-sensitivity UV absorbance detection in capillary electrophoresis. Analytical

710

Chemistry 1993, 65 (23), 3454-3459.

711

18.

712

of specific peptides on heat-induced aggregation of β-lactoglobulin. Biomacromolecules

713

2011, 12 (6), 2159-2170.

714

19.

715

Bulletin de la Société de chimie biologique 1953, 35 (1-2), 100-116.

716

20.

717

protein structure. Faraday Symposia of the Chemical Society 1982, 17 (0), 109-120.

718

21.

719

Lactoglobulin hydrolysis. 2. Peptide identification, SH/SS exchange, and functional properties

720

of hydrolysate fractions formed by the action of plasmin. 1999, 47 (8), 2980-2990.

721

22.

722

hydrolysis and DPPH radical scavenging activity of whey protein hydrolysates. Current

723

Research in Dairy Sciences 2011, 3, 25-35.

724

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725

temperature and pH on the selective hydrolysis of whey proteins by trypsin and potential

726

recovery of native alpha-lactalbumin. International Dairy Journal 2011, 21 (3), 166-171.

Kuipers, B. J. H.; Gruppen, H., Prediction of molar extinction coefficients of proteins

Bruin, G. J. M.; Stegeman, G.; Van Asten, A. C.; Xu, X.; Kraak, J. C.; Poppe, H.,

Chervet, J. P.; Van Soest, R. E. J.; Ursem, M., Z-shaped flow cell for UV detection in

Moring, S. E.; Reel, R. T.; van Soest, R. E. J., Optical improvements of a Z-shaped cell

Kosters, H. A.; Wierenga, P. A.; de Vries, R.; Gruppen, H., Characteristics and effects

Linderstrøm-Lang, K., The initial phases of the enzymatic degradation of proteins.

Eisenberg, D.; Weiss, R. M.; Terwilliger, T. C.; Wilcox, W., Hydrophobic moments and

Caessens, P. W. J. R.; Daamen, W. F.; Gruppen, H.; Visser, S.; Voragen, A. G. J., β-

Kamau, S. M.; Lu, R.-R., The effect of enzymes and hydrolysis conditions on degree of

Cheison, S. C.; Leeb, E.; Toro-Sierra, J.; Kulozik, U., Influence of hydrolysis


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24.

Panyam, D.; Kilara, A., Emulsifying peptides from the tryptic hydrolysis of casein.

728

Journal of Food Science 2004, 69 (3), FCT154-FCT163.

729

25.

730

the pH of hydrolysis on enzyme selectivity of Bacillus licheniformis protease towards whey

731

protein isolate. International Dairy Journal 2015, 44, 44-53.

732

26.

733

substrate concentration on enzyme selectivity using whey protein isolate and Bacillus

734

licheniformis protease. Journal of Agricultural and Food Chemistry 2014, 62 (42), 10230-

735

10239.

736

27.

737

enzymatic breakdown of peptides during enzymatic protein hydrolysis. Biochimica et

738

Biophysica Acta - Proteins and Proteomics 2015, 1854 (8), 987-994.

739

28.

740

proteolysis. Part 2. Substrate regulation of peptide bond demasking and hydrolysis. Liquid

741

chromatography of hydrolyzates. Die Nahrung 1986, 30 (10), 995-1001.

742

29.

743

kinetics of peptide bond cleavage for whey protein isolate hydrolysed by Bacillus

744

licheniformis protease. Journal of Molecular Catalysis B: Enzymatic 2016, 133 (Supplement 1),

745

S426-S431.

Butré, C. I.; Sforza, S.; Wierenga, P. A.; Gruppen, H., Determination of the influence of

Butré, C. I.; Sforza, S.; Gruppen, H.; Wierenga, P. A., Determination of the influence of

Butré, C. I.; Buhler, S.; Sforza, S.; Gruppen, H.; Wierenga, P. A., Spontaneous, non-

Vorob'ev, M. M.; Paskonova, E. A.; Vitt, S. V.; Belikov, V. M., Kinetic description of

Vorob’ev, M. M.; Butré, C. I.; Sforza, S.; Wierenga, P. A.; Gruppen, H., Demasking

746



Page 34 of 52

Figure legends Table 1: Selectivity rules for bovine trypsin 2 and the number of correctly predicted cleavage sites (CSs) of the total CSs in α-lactalbumin and β-casein for bovine, porcine and human trypsins based on the rules*.

indicates charged amino acids (KRDE) and

indicates

neutral amino acids (All others). The letters in the columns represent the abbreviation of the amino acids. “-” means there is no amino acid present.

Table 2: The nitrogen to protein conversion factor (N-factor), the protein content and purity, the molar weight, the number of peptide bonds per gram of protein (htot), the number of amino acids (#AA) and the molar extinction coefficient at 214 nm (ε214) of the three trypsins (bovine, porcine and human trypsins) and two proteins (α-lactalbumin and β-casein) used, and the number of cleavage sites (#CS), the theoretical maximum degree of hydrolysis (DHmax,theo) and the number of unique peptides annotated in each hydrolysates.

Table 3: The amino acid, peptide and molar sequence coverages from the analysis of the bovine, porcine or human tryptic hydrolysates of α-LA and β-cas.

Table 4: Individual and total hydrolysis rate constants (±16 % standard error) of all cleavage sites (CSs) in α-LA during hydrolysis by bovine, porcine or human trypsins based on the CS products concentrations. CSs classified as high, intermediate and low selectivity sites were highlighted with , and , respectively.

Table 5: Individual and total hydrolysis rate constants (±15 % standard error) of all cleavage sites (CSs) in β-cas during hydrolysis by bovine, porcine or human trypsins based on the CS 34 ACS Paragon Plus Environment

Page 35 of 52


products concentrations. CSs classified as high, intermediate and low selectivity sites were highlighted with , and , respectively.

Table 6: Number of cleavage sites in α-LA and β-cas towards which bovine, porcine and human trypsins have high, intermediate or low/zero selectivity.

Table 7: Porcine trypsin hydrolysis rate constants and the amino acid sequences of binding site positions (from P4 to P4’) of cleavage sites in α-LA and β-cas. Amino acids with a negatively charged side chain (DE) are marked with . Amino acids with a positively charged side chain (KR) are marked with

except when they are on P1. Cysteines are marked with .

“-” means there is no amino acid present. The

emphasized the P2 position of the binding

site.

Table 8: Human trypsin hydrolysis rate constants and the amino acid sequences of binding site positions (from P4 to P4’) of cleavage sites in α-LA and β-cas. Amino acids with a negatively charged side chain (DE) are marked with . Amino acids with a positively charged side chain (KR) are marked with



emphasized the P2 and P1’ positions of the

binding site.

Table 9: Revised rules for porcine and human trypsin selectivity towards cleavage sites (CSs) in a protein, and the number of correctly predicted CSs of the total CSs in α-LA and β-cas based on the rules.

indicates negatively charged amino acids (DE) and

indicates all other

amino acids. The letters in the columns represent the abbreviation of the amino acids. 35 ACS Paragon Plus Environment


Figure 1: Hydrolysis curves (DHstat versus time) of hydrolysis of (A) α-LA and (B) β-cas by (—) bovine, (—·) porcine or (---) human trypsins. The dotted lines (······) indicate the DHmax,theo.

Figure 2: Percentage of remaining intact (A, B) α-LA and (C, D) β-cas in the hydrolysates during hydrolysis by ( ) bovine, () porcine or () human trypsins as a function of (A, C) time and (B, D) DHstat/DHmax,fit. The lines in panel (B) and (D) were used to guide the eye. The error bars were smaller than the marker size.

Figure 3: Comparison of DH calculated based on peptide analysis (DHMS) and pH-stat titration (DHstat) for (A) α-LA and (B) β-cas hydrolysis by ( ) bovine, () porcine or () human trypsins. The dotted line (······) indicates the function y=x. The error bars are typically smaller than the marker size.

Figure 4: Concentrations of peptides (A) α-LA[14-16] and (B) α-LA[1-10], cleavage site products (C) α-LA K13-14 and (D) α-LA R10-11 formed during hydrolysis by ( ) bovine, () porcine or () human trypsins. The dotted lines (······) represent the initial injected concentration of α-LA. In panels (C) and (D), the dashed lines (------) represent the fits using the 2nd order reaction equation. The markers represent averaged values of duplicate hydrolysates.

Figure 5: Concentrations of peptides (A) β-cas[108-113], (B) β-cas[184-209], cleavage site products (C) β-cas K113-114, (D) β-cas K183-184 formed during hydrolysis by ( ) bovine, () porcine and ( ) human trypsins. The dashed lines (------) represent the fits using the 36 ACS Paragon Plus Environment

Page 36 of 52

Page 37 of 52


equation of 2nd order reaction and the dotted lines (······) represent the injected concentration of each protein. The inset in panel (C) shows the zoomed curve of the cleavage site products formation of β-cas K113-114 from 0-500 s. The markers are the averaged values of duplicate hydrolysates.

Figure 6: (A, C) Bovine (), porcine () and human () trypsin selectivity towards cleavage sites calculated based on the concentration of the CS products and (B, D) the total hydrolysis rate constants (∑(k R × C)) in (A, B) α-LA and (C, D) β-cas.

Figure 7: Experimental maximum degree of hydrolysis (DHmax,exp, marked with ) and the predicted maximum degree of hydrolysis based on enzyme selectivity (DHmax,pre, marked with ) of α-LA and β-cas hydrolysis by bovine, porcine or human trypsins. The dashed lines (------) represent the theoretical maximum degree of hydrolysis based on trypsin specificity (DHmax,theo) of α-LA and β-cas.



Supporting information: Supporting information for review only: written statement of the manuscript’s significance.

Supporting information for publication: Appendix A: List of peptides derived from α-LA hydrolysed by the three trypsins. (* indicates methionine oxidation)

Appendix B: List of peptides derived from β-cas hydrolysed by the three trypsins. (* indicates phosphoserine)

Appendix C: Molar sequence coverages of α-LA hydrolysates catalysed by (A) bovine, (B) porcine or (C) human trypsins at all DH values. The dotted line (······) indicates the initial protein concentration.

Appendix D: Molar sequence coverages of β-cas hydrolysates catalysed by (A) bovine, (B) porcine or (C) human trypsins at all DH values. The dotted line (······) indicates the initial protein concentration.

Appendix E: List of peptide concentrations at all DH values in α-LA hydrolysed by porcine or human trypsins

Appendix F: List of peptide concentrations at all DH values in β-cas hydrolysed by porcine or human trypsins


Page 38 of 52

Page 39 of 52


Table 1: Selectivity rules for bovine trypsin 2 and the number of correctly predicted cleavage sites (CSs) of the total CSs in α-lactalbumin and β-casein for bovine, porcine and human trypsins based on the rules*.

indicates charged amino acids (KRDE) and

indicates

neutral amino acids (All others). The letters in the columns represent the abbreviation of the amino acids. “-” means there is no amino acid present. Binding site P2 P1 P1’ P2’ High selectivity All others K/R All others Intermediate KRDE K/R All others selectivity All others K/R KRDE KRDE K/R KRDE Low/zero K/R selectivity K/R P C K/R Category

Number of correctly predicted CSs/total CSs Bovine trypsin Porcine trypsin Human trypsin 11/12 14/21 9/14 5/9

0/2

2/3

1/7 2/7

2/5

3/11

1/7

* To avoid repetition, the right part of the table (number of correctly predicted CSs/total CSs) was from experimental data, which will be discussed in section 3.5.



Page 40 of 52

Table 4.2: The nitrogen to protein conversion factor (N-factor), the protein content and purity, the molar weight, the number of peptide bonds per gram of protein (htot), the number of amino acids (#AA) and the molar extinction coefficient at 214 nm (ε214) of the three trypsins (bovine, porcine and human trypsins) and two proteins (α-lactalbumin and β-casein) used, and the number of cleavage sites (#CS), the theoretical maximum degree of hydrolysis (DHmax,theo) and the number of unique peptides annotated in each hydrolysates. Protein α-LA β-cas BT PT HT

Uniprot 1 code. P00711 P02666 P00760 P00761 P07478

1

N-factor [g of protein/g N] 6.25 6.39 5.97 5.84 5.98

Protein content [w/w] 93 % 90 % 80 % 86 % 77 %

2

Purity [%] 90 90 100 100 100

Molar weight [Da] 14,186

3

6

23,983 23,305 23,475 24,041

4

htot [mmol/g] 8.6 8.7 -

#CS

1

13 15 -

1

#AA

123 209 223 223 224

DHmax,theo [%] 10.7 7.2 -

Ɛ214

-1

6

-1

[M ·cm ] 300,461 424,047 -

#β-cas peptides

23 30 30

34 36 40

α-LA: α-lactalbumin; β-cas: β-casein; BT: bovine trypsin; PT: porcine trypsin; HT: human trypsin 1

From Uniprot (http://www.uniprot.org)

2

The protein purity is defined as the percentage of UV280 peak area of the target protein out of total UV280 area from RP-UPLC.

3

The molar weight was calculated with 5 phosphoserines on the β-cas molecule as shown in RP-UPLC-MS.

4

htot is the number of millimoles of peptide bonds per gram of protein calculated using the amino acid sequence and molecular weight of the protein reported in Uniprot.

6,7

#α-LA and β-cas peptides stand for the unique peptides identified in the hydrolysates of α-LA and β-cas, respectively.

40

ACS Paragon Plus Environment

7

#α-LA peptides

Page 41 of 52


Table 3: The amino acid, peptide and molar sequence coverages from the analysis of the bovine, porcine or human tryptic hydrolysates of α-LA and β-cas. Average sequence coverage [%] Amino acid Peptide Molar Protein α-LA β-cas α-LA β-cas α-LA Enzyme Bovine 100±0 100±0 96±3 83±10 80±9 Porcine 100±0 99.5±1 90±2 87±8 70±11 Human 100±0 100±0 95±5 91±4 78±8

β-cas 74±8 72±7 81±8

Table 4: Individual and total hydrolysis rate constants (±16 % standard error) of all cleavage sites (CSs) in α-LA during hydrolysis by bovine, porcine or human trypsins based on the CS products concentrations. CSs classified as high, intermediate and low selectivity sites were highlighted with , and , respectively. Hydrolysis rate constants , -3 [10 /s/mgenzyme] Bovine Porcine Human trypsin trypsin trypsin 0.5(±0.1) 3.4(±0.1) 1.3(±0.2) 3.0(±0.0) 2.8(±0.2) 2.7(±0.1) 2.3(±0.0) 2.4(±0.2) 0.2(±0.0) 1.2(±0.0) 3.4(±0.1) 1.3(±0.2) 1.3(±0.2) 1.8(±0.3) 2.2(±0.5) 0.0(±0.0) 1.7(±0.1) 0.07(±0.00) 0.05(±0.01) 0.01(±0.00) 0.3(±0.1) 0.05(±0.01) 2.1(±0.0) 0.3(±0.1) 0.4(±0.0) 2.3(±0.0) 1.6(±0.5) 0.5(±0.0) 1.5(±0.2) 0.9(±0.1) 1.3(±0.0) 3.9(±0.4) 1.2(±0.0) 0.3(±0.1) 0.004(±0.000) 0.004(±0.000) 0.0(±0.0) 0.002(±0.000) 0.002(±0.000) 10.9 25.3 12.1 ¡¢£¤¥¡,¦§¨

Enzyme CS K5 R10 K13 K16 K58 K62 K79 K93 K94 K98 K108 K114 K122 Total



Page 42 of 52

Table 5: Individual and total hydrolysis rate constants (±15 % standard error) of all cleavage sites (CSs) in β-cas during hydrolysis by bovine, porcine or human trypsins based on the CS products concentrations. CSs classified as high, intermediate and low selectivity sites were highlighted with , and , respectively. Hydrolysis rate constants ,

¡¢£¤¥¡,© ª«

[10

Enzyme CS R1 R25 K28 K29 K32 K48 K97 K99 K105 K107 K113 K169 K176 R183 R202 Total

Bovine trypsin 0.0(±0.0) 1.4(±0.4) 16.7(±0.9) 1.8(±0.1) 1.6(±0.7) 0.002(±0.000) 1.3(±0.3) 31.4(±1.9) 47.8(±3.5) 5.7(±2.0) 2.3(±0.2) 66.4(±8.2) 23.8(±7.1) 6.0(±1.9) 0.4(±0.2) 207.6

-3

/s/mgenzyme]

Porcine trypsin 0.0(±0.0) 14.1(±1.3) 114.8(±21.9) 4.4(±0.3) 8.0(±0.14) 0.2(±0.0) 53.1(±3.7) 17.5(±0.8) 92.6(±20.1) 5.9(±1.8) 10.1(±0.7) 191.5(±13.1) 98.7(±27.8) 37.8(±2.8) 2.5(±0.3) 651.2

Human trypsin 0.0(±0.0) 64.4(±7.6) 56.2(±2.6) 0.1(±0.0) 0.9(±0.1) 0.7(±0.1) 53.7(±2.6) 0.5(±0.0) 12.1(±0.3) 0.7(±0.2) 0.1(±0.0) 19.4(±0.7) 13.9(±1.1) 10.2(±0.1) 0.04(±0.0) 232.9

Table 6: Number of cleavage sites in α-LA and β-cas towards which bovine, porcine and human trypsins have high, intermediate or low/zero selectivity.

Enzyme Category High selectivity sites Intermediate selectivity sites Low/zero selectivity sites

Bovine trypsin 5 4 4

α-LA Porcine trypsin 10 3

Human trypsin 6 3 4


Bovine trypsin 7 5 3

β-cas Porcine trypsin 11 2 2

Human trypsin 7 1 7

Page 43 of 52


Table 7: Porcine trypsin hydrolysis rate constants and the amino acid sequences of binding site positions (from P4 to P4’) of cleavage sites in α-LA and β-cas. Amino acids with a negatively charged side chain (DE) are marked with . Amino acids with a positively charged side chain (KR) are marked with



emphasized the P2 position of the binding

site. Cleavage site

¬ ,

Binding site (1/s/mgenzyme) P4 P3 P2 P1 P1' P2' P3' P4'

High selectivity α-LA K108 3.9E-3 α-LA K5 3.4E-3 α-LA K16 3.4E-3 α-LA R10 2.8E-3 α-LA K13 2.4E-3 α-LA K94 2.3E-3 α-LA K93 2.1E-3 α-LA K58 1.8E-3 α-LA K62 1.7E-3 α-LA K98 1.5E-3 β-cas K169 1.9E-1 β-cas K28 1.1E-1 β-cas K176 9.9E-2 β-cas K105 9.3E-2 β-cas K97 5.3E-2 β-cas R183 3.8E-2 β-cas K99 1.7E-2 β-cas R25 1.4E-2 β-cas K113 1.0E-2 β-cas K32 7.9E-3 β-cas K107 5.9E-3 Intermediate selectivity β-cas K29 4.4E-3 β-cas R202# 2.5E-3 Low/zero selectivity α-LA K79 1.4E-5 α-LA K114 3.6E-6 α-LA K122 2.2E-6 β-cas K48 2E-4 β-cas R1 0 #

L A Q L K D E V R E C V M C I N I W I L S Q R I V P M A G V Y P S K S I P F K I P K

H T L F L K V N C D S N Q P S Q V T P E H

K K K R K K K K K K K K K K K R K R K K K

A C G E D I K I D V V K A H V D E I Y F E

L C S E V F Y G G L K D L K G L D K I L D W C K D Q N G I N L P V I E K V P Y K E M K E A M P I A M A N K K P V E Q S* E M P F

I G

N P

K V

K R

I G

E P

K F

F P

S C L L -

C S C Q -

D E E D -

K K K K R

F L L I E

L D H L

D Q P E

D W F E

represents masked cleavage site in intact protein

* represents phosphoserine



Page 44 of 52

Table 8: Human trypsin hydrolysis rate constants and the amino acid sequences of binding site positions (from P4 to P4’) of cleavage sites in α-LA and β-cas. Amino acids with a negatively charged side chain (DE) are marked with . Amino acids with a positively charged side chain (KR) are marked with



emphasized the P2 and P1’ positions of the

binding site. Cleavage site

® ,

Binding site (1/s/mgenzyme) P4 P3 P2 P1 P1' P2' P3' P4'

High selectivity α-LA R10 2.7E-3 α-LA K58 2.2E-3 α-LA K94 1.6E-3 α-LA K5 1.3E-3 α-LA K16 1.3E-3 α-LA K108 1.2E-3 β-cas R25 6.4E-2 β-cas K28 5.6E-2 β-cas K97 5.3E-2 β-cas K169 1.9E-2 β-cas K176 1.4E-2 β-cas K105 1.2E-2 β-cas R183 1.0E-2 Intermediate selectivity α-LA K98 8.5E-4 α-LA K79 3.2E-4 α-LA K93 3.2E-4 β-cas K32 9.0E-3 Low/zero selectivity α-LA K13 2.3E-4 α-LA K62 6.9E-5 α-LA K114 3.6E-6 α-LA K122 2.2E-6 β-cas K107 7.4E-4 β-cas K48 7.0E-4 β-cas K113# 8.0E-5 β-cas K29 7.1E-5 β-cas K99 5.1E-5 # β-cas R202 3.8E-5 β-cas R1 0 #

E I C Q K L S R G S V M Y

V N V L D A I I V Q P A P

F N K T L H T N S S Q P Q

R K K K K K R K K K K K R

E I I C G A I K V V A H D

L W L E Y L N I K L V K M

K C D V G C K E E P P E P

D K K F G S K K A V Y M I

I S M K

L C C I

D D V E

K K K K

V F K F

G I L D I L Q S*

N D D E

R I C L P L P I S G -

E W S C K Q F N K P -

L C E E H D P K V V -

K K K K K K K K K R R

D D L L E I Y I E G E

L K G D Q N D Q W - M P F H P F P V E E K F A M A P F P L E E

represents masked cleavage site in intact protein

* represents phosphoserine


Page 45 of 52


Table 9: Revised rules for porcine and human trypsin selectivity towards cleavage sites (CSs) in a protein, and the number of correctly predicted CSs of the total CSs in α-LA and β-cas based on the rules.

indicates negatively charged amino acids (DE) and

indicates all other

amino acids. The letters in the columns represent the abbreviation of the amino acids. Porcine trypsin Binding sites P2 P1 All others K/R

Enzyme Categories

High selectivity Intermediate or low selectivity Enzyme Categories

High selectivity Intermediate or low selectivity

% corrected predicted CS

19/21

K/R

DE

5/7

Total: Human trypsin Binding sites P2 P1 P1’ All others K/R All others DE K/R All others All others K/R DE DE K/R DE Total:

24/28

10/14 6/15 4/15 1/15 21/28

A

B

10 6

DHstat (%)

DHstat (%)

8 6 4

4

2 2 0

0 0

2000

4000

6000

8000

10000

0

2000

Time (s)

4000

6000

8000

10000

Time (s)

Figure 1: Hydrolysis curves (DHstat versus time) of hydrolysis of (A) α-LA and (B) β-cas by (—) bovine, (—·) porcine or (---) human trypsins. The dotted lines (······) indicate the DHmax,theo.



A

80

60

40

20

B

100

% remaining intact α-LA

% remaining intact α-LA

100

0

80 60 40 20 0

0

2000

4000

6000

8000

10000

0

20

Time (s)

40

60

80

100

DHstat/DHmax,fit (%) C

D

100

% remaining intact β-cas

100

% remaining intacβ-cas

Page 46 of 52

80

80

60

60

40

40

20

20

0

0 0

2000

4000

6000

8000

10000

0

20

Time (s)

40

60

80

100

DHstat/DHmax,fit (%)

Figure 2: Percentage of remaining intact (A, B) α-LA and (C, D) β-cas in the hydrolysates during hydrolysis by ( ) bovine, () porcine or () human trypsins as a function of (A, C) time and (B, D) DHstat/DHmax,fit. The lines in panel (B) and (D) were used to guide the eye. The error bars were smaller than the marker size.


Page 47 of 52


B

A

10

8

8

DHMS (%)

DHMS (%)

6 6 4

4

2

2 0

0 0

2

4

6

8

10

0

2

4

6

8

DHstat (%)

DHstat (%)

Figure 3: Comparison of DH calculated based on peptide analysis (DHMS) and pH-stat titration (DHstat) for (A) α-LA and (B) β-cas hydrolysis by ( ) bovine, () porcine or () human trypsins. The dotted line (······) indicates the function y=x. The error bars are typically smaller than the marker size.



Page 48 of 52

A

B 60

Peptide concentration (µM)


60 50 40 30 20 10

40 30 20 10 0

0 0

2000

4000

6000

8000

0

10000

Time (s)

2000

C

60 50 40 30 20 10 0

4000

6000

8000

10000

Time (s)

CS product concentration (µM)


50

D

60 50 40 30 20 10 0

0

2000

4000

6000

8000

10000

0

2000

4000

6000

8000

10000

Time (s)

Time (s)

Figure 4: Concentrations of peptides (A) α-LA[14-16] and (B) α-LA[1-10], cleavage site products (C) α-LA K13-14 and (D) α-LA R10-11 formed during hydrolysis by ( ) bovine, () porcine or () human trypsins. The dotted lines (······) represent the initial injected concentration of α-LA. In panels (C) and (D), the dashed lines (------) represent the fits using the 2nd order reaction equation. The markers represent averaged values of duplicate hydrolysates.


Page 49 of 52


AB Peptide concentration (µM)


40

30

20

10

0

DB

40

30

20

10

0 0

8000

1000

10000

0

8000

1000

Time (s)

10

30

20

10

0 0

200

400

Time (s)

D CS product concentration (µM)

30 Concentration (µM)


C

20

10000

Time (s)

0

30

20

10

0 0

2000

4000

6000

8000

10000

0

2000

Time (s)

4000

6000

8000

10000

Time (s)

Figure 5: Concentrations of peptides (A) β-cas[108-113], (B) β-cas[184-209], cleavage site products (C) β-cas K113-114, (D) β-cas K183-184 formed during hydrolysis by ( ) bovine, () porcine and ( ) human trypsins. The dashed lines (------) represent the fits using the equation of 2nd order reaction and the dotted lines (······) represent the injected concentration of each protein. The inset in panel (C) shows the zoomed curve of the cleavage site products formation of β-cas K113-114 from 0-500 s. The markers are the averaged values of duplicate hydrolysates.



Page 50 of 52

A

Selectivityα-LA (%)

25 20 15 10 5

30

Total hydrolysis rate constant (10-3/s/mgenzyme)

30

0

B

25 20 15 10 5 0

K5

R10

K13

K16

K58

K62

K79

K93

K94

K98

K108

K114

K122

C

35

Selectivityβ-cas (%)

30 25 20 15 10 5 0

Total hydrolysis rate constant (10-3/s/mgenzyme)

Cleavage sites 40

700

D

600 500 400 300 200 100 0

R1

R25

K28

K29

K32

K48

K97

K99

K105 K107 K113 K169 K176 R183 R202

Cleavage sites

Figure 6: (A, C) Bovine (), porcine () and human () trypsin selectivity towards cleavage sites calculated based on the concentration of the CS products and (B, D) the total hydrolysis rate constants (∑(k R × C)) in (A, B) α-LA and (C, D) β-cas.


Page 51 of 52


α-LA DHmax,theo 10 β-cas DHmax,theo

DHmax (%)

8 6 4 2 0

Bovine Porcine Human Bovine Porcine Human α-LA α-LA α-LA β-cas β-cas β-cas

Figure 7: Experimental maximum degree of hydrolysis (DHmax,exp, marked with ) and the predicted maximum degree of hydrolysis based on enzyme selectivity (DHmax,pre, marked with ) of α-LA and β-cas hydrolysis by bovine, porcine or human trypsins. The dashed lines (------) represent the theoretical maximum degree of hydrolysis based on trypsin specificity (DHmax,theo) of α-LA and β-cas.



For Table of Contents Only


Page 52 of 52

Comparison of Protein Hydrolysis Catalyzed by Bovine, Porcine, and

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