Optimization of Amino Acid Derivatization after ... - ACS Publications

Subscriber access provided by UNIV OF SOUTHERN INDIANA

New Analytical Methods

Total Protein Analysis in Algae via Bulk Amino Acid Detection: Optimization of Amino Acid Derivatization after Hydrolysis with O-Phthalaldehyde 3-Mercaptopropionic Acid (OPA-3MPA) Hunter Cuchiaro, and Lieve M. L. Laurens J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00884 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on May 5, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

Total Protein Analysis in Algae via Bulk Amino Acid Detection: Optimization of Amino Acid Derivatization after Hydrolysis with O-Phthalaldehyde 3-Mercaptopropionic Acid (OPA3MPA)

Hunter Cuchiaro§, and Lieve M.L. Laurens* National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado, USA * Author for correspondence; Phone: +1 (303) 384-6196; email: [email protected] § current

address: 1301 Center Ave, Colorado State University, Fort Collins, CO 80523

ACS Paragon Plus Environment


1

Abstract

2

The analysis of protein in algal biomass is one of the most critical areas of commercial

3

development of algae characterization for nutritional or other high value applications. A new

4

rapid and accurate method is required that can be widely implemented and that is free of

5

interferences from the complex algal biomass matrix. We developed a simple spectrophotometric

6

method for primary amino acid quantification bulk measurement in an acid hydrolyzed algal

7

biomass preparation, as an alternative to the more labor-intensive amino HPLC acid analysis or

8

less specific nitrogen-to-protein conversion. We have validated an O-phthalaldehyde (OPA)-

9

based derivatization method, showing accurate and linear quantification for standard reference

10

amino acids as well as mixtures, mimicking the amino acid complexity found in algal biomass.

11

The presence of interferences that may be derived from the complex biomass biochemical

12

composition was tested during the method validation phase. We document the application of a

13

novel method of OPA derivatization with 3-mercaptopropionic acid (MPA) to determine the total

14

amino acid content of harvested algal biomass collected from different, controlled cultivation

15

conditions and demonstrated a within 10% accuracy against a reference measurement of amino

16

acid content in at least 4 species and 10 algal biomass samples, across early, mid and late-stage

17

of cultivation.

18 19

Acronyms

20

IEC – Ion Exchange Chromatography; RP-HPLC – Reverse Phase High Performance Liquid

21

Chromatography; OPA – O-phthalaldehyde; 2ME – 2-mercaptoethanol; 3MPA – 3-

22

mercaptopropionic acid; AA – amino acid; NPN – Non-protein nitrogen.

23


Page 2 of 30

Page 3 of 30


24

Keywords

25

Amino Acid Analysis; Algae; OPA; o-phthalaldehyde; Spectrophotometry.

26 27 28 29

Background The protein content of algae has a strong influence on determining the economic potential

30

for food and feed uses for algal biomass and thus accurately determining protein content is

31

critically important. The protein content of microalgae can range from 7% to 40% and can

32

change dramatically over the course of its lifecycle, however, the biochemical composition of the

33

biomass is highly complex and thus many analytical methods suffer significant interferences

34

when characterizing different individual constituents.1–6 For example, it is known that there are

35

significant interferences from reducing substances, such as sugars, on the color-development of

36

the bicinchoninic acid (BCA) colorimetric protein method based on Lowry’s procedure.7,8 The

37

presence of reducing sugars in the hydrolyzed whole biomass used for protein determination is

38

thought to be responsible for an up to two-fold overestimation of protein content when compared

39

against total amino acid content of the same biomass.8 Those spectrophotometric methods

40

referred to as the Lowry et al. or Bradford are also of limited utility unless used to quantify

41

purified protein, as measured by dry weight estimation.7,9,10

42 43

Total protein content can be determined via a nitrogen-to-protein conversion factor,

44

which is typically derived from the respective amino acid composition of algal biomass.11 Amino

45

acid analysis is a common analysis when characterizing biomass and is usually done by the use

46

of ion exchange chromatography (IEC) or reverse phase liquid chromatography (RP-HPLC)



47

followed by a post-column ninhydrin derivatization. Ninhydrin reacts with the sample and the

48

mixture is heated to 100 ºC, and the resulting Ruhemann’s purple product is thereby measured

49

colorimetrically.12–16 This established method can be applied to separations or bulk materials, but

50

high oxygen and light sensitivity, slow reaction time at high temperature, and high background

51

absorbance are disadvantageous, as is the need for specialized and dedicated instrument set up.17

52

Ninhydrin is also known to react with imines, guanidino groups (found in arginine), amides

53

(found in asparagine and glutamine), indole rings (tryptophan), sulfhydryl groups (cysteine),

54

cytosine, guanine, and cyanide ions to form chromophores and thus linking ninhydrin

55

derivatization detection with HPLC separation is appropriate to reduce these interferences.16

56

Existing methodologies based on combustion (e.g. Dumas method) are widely used to

57

determine protein but often rely on a dedicated nitrogen conversion factors, based on the

58

nitrogen content of each amino acid.11,18 Amino acid analysis can be complex, labor intensive

59

and is sometimes out of reach for rapid characterization of biomass to laboratories that are not

60

equipped with complex instrumentation, such as the HPLC instrumentation to support the

61

analysis of individual amino acids. Amino acid derivatization itself can be performed easily at

62

mild temperatures with appropriate derivatization agents and as long as no other products that are

63

present interfere with the colorimetric reaction.

64

In the early 1970’s a method based on O-phthalaldehyde (OPA) and 2-mercaptoethanol

65

(2ME) was developed as an alternative method for amino acid (AA) detection. The fluorescent

66

adduct was specific to primary amines.19,20 The greater specificity of the OPA/thiol/AA adduct is

67

advantageous over ninhydrin when considering complex samples, by simplifying sample

68

preparation.21 Additionally, proline, with its unique pyrrolidinyl structure, can be measured after

69

perchloric acid pre-treatment.22,23 The primary disadvantage of this method was that the analyte


Page 4 of 30

Page 5 of 30


70

is an intermediate that decays to a non-productive final product.24 Much work has been done to

71

study the kinetics of this reaction, and it is often stated in the literature that measurements should

72

be performed at the same timepoint to preserve accuracy.25–27 The adduct is stabilized by

73

substituting Roth’s original 2ME for 3-mercaptopropionic acid (3MPA), reaction mechanism is

74

shown in Figure 1.28

75

A review of the literature revealed that no study had been carried out using OPA/3MPA

76

derivative absorbance to quantify total amino acids in a bulk preparation without the

77

implementation of a liquid chromatography.29–34 The objective of this work was to develop and

78

optimize a simple spectrophotometric method for the bulk determination of amino acids in algal

79

biomass as an approximation of total protein content. UV/Vis spectrophotometry is a widely

80

available, low-cost technique that is well suited to high-throughput analysis. The absorbance of

81

OPA/AA derivatives could be quantified using a summation of relative absorbance response

82

factors.35 When comparing amino acid profiles across 14 species and growth conditions,

83

previous research in our group found that the normalized weight percentage of amino acids was

84

relatively stable.11 Arginine, lysine, and alanine had the greatest range in normalized weight

85

percentage.11 It was hypothesized that variations in individual amino acid content between

86

species and growth condition would not substantially affect the overall absorptivity of analytical

87

mixtures and thus a bulk measurement based on amino acid derivatization may prove a

88

promising approach.

89

The objectives of this work were to optimize a method for OPA/AA derivatization,

90

accurately measure amino acid content in bulk amino acids standards, investigate the impact of

91

interfering compounds within process streams, and to compare this new method’s data accuracy



92

on hydrolyzed protein from biomass against total amino acid data obtained from a commercial

93

analytical laboratory.

94 95

Materials and Methods

96

Materials

97

O-phthalaldehyde (P0657), 3-mercaptopropionic acid (M5801), L-arginine (A5006), L-

98

aspartic acid (A9256), L-alanine (A7627), L-cysteine (C7352), L-glutamic acid (G1251), glycine

99

(G8898), L-histidine (H8000), L-isoleucine (I2752), L-leucine (L8000), L-lysine (L5501), L-

100

methionine (M9625), L-phenylalanine (P2126), L-serine (S4500), L-threonine (T8625), L-

101

tryptophan (T0254), L-tyrosine (T3754), L-valine (V0500) were purchased from Sigma Aldrich.

102

For all solution, nanopure water was used (18 MΩ deionized water). Methanol (A412P-4),

103

sodium chloride (S271-500), ethanol (A4094), boric acid (A73-1), sodium nitrate (S343-500),

104

ammonium nitrate (A676-500), sodium hydroxide (SS256-500), guanidinium hydrochloride

105

(AAJ7582322) were purchased from Fisher Scientific. All reagents were ACS-grade or better.

106

Absorbance measurements were taken using a Beckman Coulter DV 800

107

spectrophotometer, equipped with a six-position automated cell holder, and 10mm pathlength,

108

1.0 mL matched quartz cuvettes (50-823-023) purchased from Fisher Scientific. Single-

109

wavelength absorbance measurements were made at 334 nm and absorbance spectra recorded

110

between 600-250 nm.

111 112 113 114

Solutions Preparation Diluent (20% 0.2M Sodium tetraborate, pH 9.3 / 80% Methanol) was prepared by dissolving 6 g boric acid in 480 mL water, and pH was then adjusted to 9.3 +/-0.5 with 5 M


Page 6 of 30

Page 7 of 30


115

NaOH. 400 mL of this solution was combined with 1600 mL methanol and stirred until the

116

solution returned to room temperature. The prepared solution was stored at room temperature in

117

a flammables cabinet.

118 119 120

OPA/3MPA Reagent Preparation OPA was prepared to a final concentration of 8mM in water and sonicated to dissolve. To

121

the same flask, 3MPA was added so that the final concentration would be 24 mM. OPA/3MPA

122

reagent was prepared fresh daily.

123 124 125

Individual Amino Acid Stock Stock solutions of each amino acid (L-Aspartic Acid, L-Glutamic Acid, L-Threonine,

126

L-Serine, L-Glycine, L-Alanine, L-Valine, L-Methionine, L-Leucine, L-Isoleucine, L-

127

Tyrosine, L-Phenylalanine, L-Tryptophan, L-Lysine, L-Histidine, L-Arginine, L-Cysteine)

128

were prepared at a concentration of 2mM in diluent using volumetric glassware. Solutions were

129

stored at 5 ºC and used within two weeks of preparation.

130 131

Bulk Amino Acid Stock Preparation

132

When preparing bulk stock solutions, amino acids were divided into groups of four (A:

133

L-Aspartic Acid, L-Threonine, L-Serine, L-Glutamate; B: L-Glycine, L-Alanine, L-Valine, L-

134

Methionine; C: L-Isoleucine, L-Leucine, L-Tyrosine, L-Phenylalanine; D: L-Tryptophan, L-

135

Lysine, L-Histidine, L-Arginine). Each bulk had concentration of 500 µM per amino acid, and

136

the total concentration of each bulk solution was 2mM total amino acids using volumetric



137

glassware. Stocks were sonicated for 10 minutes and vortexed for 30 seconds, repeated once.

138

These are the intermediate stocks.

139

Bulk analytical stock solutions were prepared by combining intermediate stocks A, B, C,

140

and D 1:2:3:4 (stock 1), 2:3:4:1 (stock 2), 3:4:1:2 (stock 3), and 4:1:2:3 (stock 4). Replicates of

141

each analytical stock were prepared together and stored at 5 °C.

142 143 144

Interference Stocks Chloride, sulfate, nitrate, ammonium, hexose sugars (1:2 mannose:glucose), ethanol and

145

guanidinium stocks were prepared at a concentration of 1.25 mM. Chloride, sulfate, nitrate,

146

ammonium, and guanidinium were dissolved in deionized water. Hexose sugars and ethanol

147

were dissolved in diluent.

148 149 150

Sample Preparation Algal samples were prepared and analyzed in triplicate for 3 species (S. obliquus, N.

151

oculata, and C. vulgaris) at early-, mid-, and late-stage growth, and D. aramatus at mid-stage

152

growth. Replicates were prepared by weighing between 10 and 15 mg (± 1.5 mg) lyophilized

153

algal biomass into Agilent borosilicate HPLC vials. For hydrolysis, 1 mL 6 N HCl was added to

154

each sample. Vials were capped with PTFE septa and vortexed for 20 seconds. Vials were

155

hydrolyzed at 110 ºC in a heating block for 24 hours, then vortexed for 20 seconds, uncapped,

156

and set to dry under nitrogen. 1 mL methanol was added to each vial and samples were again

157

vortexed for 20 seconds, then set to dry again under nitrogen. Samples were then placed under

158

vacuum at 40 °C to finish drying overnight.


Page 8 of 30

Page 9 of 30

159


Vials were returned to room temperature on benchtop (about 10 minutes). 1000 µL

160

diluent was added to each sample, then immediately recapped. Samples were reconstituted by

161

vortexing vials for 90 seconds each, then for an additional 20 seconds immediately prior to

162

filtration through 0.2 µm PTFE syringe filters into clean HPLC vials.

163

A total of 690 µL of biomass filtrate was diluted to final volume 5.0 mL in falcon tubes.

164

Tubes were sealed and then vortexed to mix. This is the analytical sample stock. BSA samples

165

were prepared using the same method, but with a dilution of 140 µL to 5mL. These are the

166

analytical stock solutions. Approximately 1.5 mL of analytical stock solution was tranferred to

167

clean, fresh HPLC vials to prepare individual replicates, capped and stored at -20 ºC.

168 169

Analytical Dilutions

170

For the individual and bulk amino acid standards, the reaction mixture consisted of a total

171

volume of 5 mL, including amino acids from the stock at concentrations between 20 and 100 µM

172

diluted with diluent, and brought to final volume with 2.5 mL of the OPA/3MPA stock solution

173

(final concentrations of 4 mM OPA and 12 mM 3MPA respectively). Samples were vortexed for

174

20 seconds after the addition of OPA/3MPA, and reaction times were measured relative to the

175

completion of this step. Measurements were corrected by subtracting the average OPA

176

interference collected over the course of the study. Interference stocks were diluted to final

177

concentrations between 5 and 125 µM in the reaction mixture, in the presence of consistently 60

178

µM amino acid bulk solution (as described above). For each of the hydrolyzed biomass samples,

179

a total of 0.25 mL of analytical stock was combined with 2.25 mL diluent and 2.5 mL

180

OPA/3MPA stock solution as described above. Measurement spectra were normalized by using

181

diluent, OPA, and sample blanks.



182 183

Results and Discussion

184

Derivatization reaction optimization

185

The derivatization reaction was optimized by measuring the absorbance of individual

186

amino acid standard solutions. A calibration curve was made by diluting stocks between 20 and

187

100 µM and measuring absorbance spectra in 15-minute intervals after the OPA/3MPA addition

188

step between 0-90 minutes. Instrument response was observed to increase from 0-30 minutes,

189

and then slowly decay from 30-90 minutes. Figure 2 shows characteristic spectra after 30-

190

minute derivatization time, with absorbance peak at 334 nm. This was the analytical wavelength

191

used for subsequent measurements. We calculated the linearity of the calibration curve at each

192

timepoint for each amino acid. We also attempted this experiment using a pH 7.2

193

phosphate/methanol (50/50 % v/v) diluent and confirmed that the lower pH is unsuitable for

194

OPA/3MPA derivatization.

195

Based on this initial study, we selected a window between 30-60 minutes for further

196

study. Fresh individual amino acid standards were prepared following the same procedure and

197

measured at 5-minute intervals from 30-55 minutes. The same general trend of signal decay was

198

observed over the course of each measurement, which we attributed to the conversion of the

199

AA/OPA/3MPA derivative to the non-absorbing final product reported in the literature.22 We

200

then prepared an OPA/3MPA blank and measured the absorbance at the same timepoints. The

201

absorbance of the OPA/3MPA blank decreased at a comparable rate as that measured in the

202

derivatized standards. When this blank absorbance was subtracted from the absorbance of the

203

derivatized standards, the rate of signal change over time was approximately 0.0003 AU/min.


Page 10 of 30

Page 11 of 30

204


We found that this trend was mostly not observed in AA solution when an OPA/3MPA

205

blank measurement was subtracted. The calibration curve slope and absorbance values remained

206

largely consistent. The average of all OPA blanks throughout this study was calculated and

207

subtracted from individual AA calibration curve measurements to refine the measurement.

208

Linear regression analysis was performed on the absorbances of each calibration curve at all

209

timepoints. The regression slopes for each amino acid were tabulated, and the correlation

210

coefficient (R2) at each timepoint averaged across 16 amino acids (Table 1). The correlation

211

coefficient (R2) was deemed suitable across all time points, ranging from 0.997 at 30 minutes to

212

0.996 at 55 minutes, with an average value of 0.997.

213

To assess the sensitivity of our method, the molar extinction coefficients for each amino

214

acid derivative were calculated at each timepoint and concentration and averaged as shown in

215

Table 2. The results of this calculation were tabulated and compared to those reported by

216

Svedas.24 The extinction coefficient for 15 of the 16 amino acids was calculated to be greater

217

than those previously reported, indicating the present method has on average 29% higher

218

sensitivity with a less than 10% relative standard deviation of the measurements, suggesting no

219

significant outliers in responses recorded. Tyrosine, the lone exception, had an extinction

220

coefficient approximately equal to the previously reported value.

221

Bulk amino acid stocks were analyzed at the same theoretical concentration as the

222

individual calibration curves. We combined the individual amino acid calibration slopes in a

223

weighted average based on the fractional concentrations within each bulk solution. This

224

calculation was performed at each timepoint and used to measure derivatized solutions’

225

absorbance from 30-55 min. The diluent blank and average OPA blank interference was

226

subtracted from measurements for each timepoint. We then calculated the AA concentration by



227

dividing the measured response by the predicted slope at each timepoint (Table 2). Measured

228

concentration was also calculated using the average normalized slope for comparison. The

229

calculated concentrations were within 4.7% of theoretical for solutions 1-3, and within 5.7% of

230

theoretical for solution 4. It was observed that using the average total predicted slope to calculate

231

each concentration produced a more precise measured value. Thus, the average predicted slope,

232

calculated across all time points, was used for subsequent measurements. Figure 3 shows results

233

from bulk solutions 1 and 3, where the concentrations of amino acid groups were inverted

234

between preparations. Linearity was preserved in both cases, demonstrating that mixtures of

235

amino acids could still be comparably measured even with differing molar absorptivities between

236

species. When we performed an ANOVA on the variance of error for each of the concentration

237

levels, we found no significant difference in variance distribution between the different

238

concentration levels and thus, with the underlying assumption of normal distribution of the

239

uncertainty, we conclude that there is no discernable trend of error with concentration.

240

It is worth noting that analysis with OPA derivatives is often done at a fixed time

241

following the introduction of OPA/3MPA reagent to sample solution. The data generated in this

242

study do not indicate a drastic change in the measured AA concentration after the subtraction of

243

the OPA blank interference over the 25-minute period presently studied.

244

The subtraction of an average OPA blank from the bulk measurement was justified by a

245

conformational study, where the bulk AA mixture was diluted at concentrations 24-96 µM

246

(inside the original curve) and an OPA blank was prepared with samples. The diluent and OPA

247

blanks were subtracted from the raw measurements, collected in triplicate, concentration

248

calculated based on the predicted slope. This procedure was repeated on three separate days. The


Page 12 of 30

Page 13 of 30


249

measurement error was not more than 6.2%, and the average measured concentrations had an R2

250

of 0.9995 across all three tests.

251

As a theoretical exercise, we modeled the expected sensitivity for each of the 14 algal

252

species/growth conditions previously reported at 30-55 minutes after derivatization.11 This was

253

done by multiplying the experimentally-derived slopes of the 16 amino acids in this study by

254

their reported concentration for in each species, and adding them to find the total. An average

255

expected slope of 0.0070 µM/AU was calculated with an RSD of 0.7% for determining the

256

amino acid concentration in algal biomass, based on these 14 species/growth conditions. This

257

was the response factor used in amino acid content calculation of algal biomass samples.

258 259 260

Limit of detection and quantification Further studies were performed using the bulk analytical standards to determine limits of

261

quantitation and detection. Experimental results were linear and met AOAC suitability percent

262

recovery criteria between 4.0 and 200 µM. The LOD and LOQ were also calculated based on the

263

average OPA blank standard deviation at each timepoint. This method produced an average LOD

264

of 2.1 µM and LOQ of 7.0 µM AA.

265 266

Interference of components in process streams

267

We assessed potential interferants that may be present during biomass processing.

268

Depending on the process stream, potentially interfering species could be introduced as reagents

269

or decomposition products. These studies included sulfate, chloride, ethanol, hexose sugars (1:2

270

mannose:glucose), nitrate, guanidinium, and ammonium, all potentially present after algal

271

biomass conversion.36 Only ammonium was predicted to interfere with AA measurement, based



272

on Roth’s original work also demonstrated reactivity of NH4+ with OPA.19 We had suspected

273

that lysine’s guanidinyl functional group had some activity with OPA, thus leading to the

274

derivative’s high molar absorptivity, the experimental data indicated that only ammonium was

275

observed to interact with the OPA (Figure 4).

276 277

Algal biomass amino acid determination

278

We tested a total of 10 algal species/growth conditions for amino acid determination in

279

this study. Scenedesmus, Nannochloropsis, and Chlorella strains were each harvested at early,

280

middle, and late growth stages, corresponding to high protein, carbohydrate, and lipid content in

281

the biomass composition. The Desmodesmus armatus strain was harvested at a middle growth

282

stage, corresponding to higher carbohydrate content. Freeze dried biomass samples were sent out

283

to a contract analytical laboratory for quantitative total amino acid analysis using the ninhydrin

284

reaction. We additionally included bovine serum albumin (BSA), a known and well-

285

characterized and purified reference protein, as a control protein sample for hydrolysis, in

286

addition to the algal biomass as a check standard and compared our results to the label claim of

287

96-99% protein. The absorbance spectra of the derivatized hydrolysis products for each of the 4

288

algae samples is presented in Figure 5.

289

We performed optimization studies on the hydrolysis method to improve hydrolysis

290

yield. We compared measured weight percent amino acid of Desmodesmus when hydrolyzed in 6

291

N HCl (control), 0.5% (w/v) phenol/6 N HCl, and 0.5% (w/v) 3MPA/6N HCl for 24 hours, and

292

also 6 N HCl for 18 hours.37 The 18 hour hydrolysis time produced a similar average

293

measurement but was less precise than the 24 hour hydrolysis time. The range between analytical

294

replicates was 25% greater for the 18 hour hydrolysis time of the same biomass. Additionally,


Page 14 of 30

Page 15 of 30


295

we tried filtering the hydrolysate and rinsing with 0.5 mL methanol prior to drying down under

296

nitrogen. None of these conditions produced a more optimal yield than the control, and in fact,

297

filtering/rinsing the hydrolysate prior to drying down effectively decreased the measured amino

298

acid yield by approximately 40%.

299

It was expected that hydrolyzed biomass would produce a systematic over-measurement

300

of protein content due to some interference from non-protein nitrogen. Ammonia and other

301

primary amines evolved from the strong acid (HCl) digestion of asparagine, glutamine, histidine,

302

tryptophan, chlorophylls, or nucleic acids could interfere with the accuracy of our method.

303

During acid hydrolysis, glutamine and asparagine are converted to glutamic acid and aspartic

304

acid respectively, and in the data obtained from our contract analytical laboratory both forms are

305

reported as a single respective measurement and thus it is impossible to estimate the contribution

306

of ammonium released from acid hydrolysis.

307

It is difficult to quantifiably estimate the primary mechanism of non-protein nitrogen

308

interference in this method. To account for any ammonium released by acid digestion of

309

nitrogen-containing amino acids, which is not typically reported by a contract analytical

310

laboratory, we applied a normalization factor based on the BSA control. Using this

311

normalization factor, our method’s over-estimation compared to contract lab analysis was

312

reduced to 11.2%. A BSA digestion was performed with each acid hydrolysis and applied to

313

those samples respectively. The BSA normalization factor was not averaged across multiple

314

analyses. Alternatively, we also considered a normalization scheme to account for ammonia

315

released from acid digestion of non-protein nitrogen (NPN) sources. We averaged 21 NPN

316

measurements previously reported in the literature and estimated an average level of 40.6% by

317

weight NPN across multiple species/timepoints.11 Since some of the NPN would manifest as



318

ammonia and some as secondary amines, nitrate or guanidine, we made the conservative

319

estimate that half of the NPN would interfere with our measurement. This 20.3% NPN correction

320

produces a more accurate measurement of the overall protein content compared to contract

321

laboratory results (average 7.9% over-estimation) but does not account for ammonia released

322

from protein decomposition. It is likely that ammoniac interference is the result of some

323

combination of protein degradation products and NPN from the biomass. Further optimization

324

studies may be warranted to determine the contribution specific to each factor (Figure 6).

325

This measurement assumes total amino acid recovery from 6 N HCl hydrolysis, and the

326

calibration curve was constructed based on 16 amino acids. These assumptions were made to

327

model the biomass hydrolysate but may contribute to the overestimation of protein content in the

328

sample.

329

Our method utilizes a simple procedure for sample preparation and analysis of protein

330

content in freeze-dried algal biomass with high sensitivity and precision, across multiple phyla

331

and 10 species/growth conditions. Analyses of bulk mixtures of standards indicates high

332

accuracy and linearity of our method from 8-200 µM amino acids. Corrections for non-protein

333

nitrogen and in particular ammonia interference in the biomass hydrolysates is necessary,

334

however this method is suitable for the rapid estimation of the bulk protein content in algal

335

biomass. Both analytical and computational suggestions are made for further optimization of this

336

analysis in future studies.

337 338

Acknowledgements

339

We would like to acknowledge Nick Sweeney and Stefanie Van Wychen for helpful discussions,

340

as well as the NREL Research Participant Graduate Internship program for making this project


Page 16 of 30

Page 17 of 30


341

possible. This work was authored by the National Renewable Energy Laboratory and financially

342

supported by the U.S. Department of Energy under Contract No. DE-AC36-08GO28308 with the

343

National Renewable Energy Laboratory, as part of the DOE Office of Energy Efficiency and

344

Renewable Energy, Bioenergy Technologies Office. The views expressed in the article do not

345

necessarily represent the views of the DOE or the U.S. Government. The U.S. Government

346

retains and the publisher, by accepting the article for publication, acknowledges that the U.S.

347

Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or

348

reproduce the published form of this work, or allow others to do so, for U.S. Government

349

purposes.

350 References (1)

Pignolet, O.; Jubeau, S.; Vaca-Garcia, C.; Michaud, P. Highly Valuable Microalgae: Biochemical and Topological Aspects. J. Ind. Microbiol. Biotechnol. 2013, 40 (8), 781– 796.

(2)

Becker, E. W. Micro-Algae as a Source of Protein. Biotechnol. Adv. 2007, 25 (2), 207– 210.

(3)

Lourenço, S. O.; Barbarino, E.; Lavín, P. L.; Lanfer Marquez, U. M.; Aidar, E. Distribution of Intracellular Nitrogen in Marine Microalgae: Calculation of New Nitrogento-Protein Conversion Factors. Eur. J. Phycol. 2004, 39 (1), 17–32.

(4)

Lourenço, S. O.; Barbarino, E. Distribution of Intracellular Nitrogen In Marine Microalgae: Basis For the Calculation of Specific Nitrogen-to-Protein Conversion Factors. J. Phycol. 1998, 34, 798–811.

(5)

Koru, E. Earth Food Spirulina (Arthrospira): Production and Quality Standards, Food



Additive; El-Samragy, Y., Ed.; Intech, 2012. (6)

Gerschwin, M. E.; Belay, A. Spirulina in Human Nutrition and Health; Gerschwin, M. E., Belay, A., Eds.; CRC Press LLC: Boca Raton, FL, 2007.

(7)

Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem. 1951, 193 (1), 265–275.

(8)

Laurens, L. M. L.; Van Wychen, S.; McAllister, J. P.; Arrowsmith, S.; Dempster, T. a; McGowen, J.; Pienkos, P. T. Strain, Biochemistry, and Cultivation-Dependent Measurement Variability of Algal Biomass Composition. Anal. Biochem. 2014, 452, 86– 95.

(9)

Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72 (1–2), 248–254.

(10)

Lucarini, A. C.; Kilikian, B. V. Comparative Study of Lowry and Bradford Methods: Interfering Substances. Biotechnol. Tech. 1999, 13 (2), 149–154.

(11)

Templeton, D. W.; Laurens, L. M. L. Nitrogen-to-Protein Conversion Factors Revisited for Applications of Microalgal Biomass Conversion to Food, Feed and Fuel. Algal Res. 2015, 11, 359–367.

(12)

Cunico, R. L.; Schlabach, T. Comparison of Ninhydrin and O-Phthalaldehyde PostColumn Detection Techniques for High-Performance Liquid Chromatography of Free Amino Acids. J. Chromatogr. 1983, 266, 461–470.

(13)

Petraco, N. D. K.; Proni, G.; Jackiw, J. J.; Sapse, A. M. Amino Acid Alanine Reactivity with the Fingerprint Reagent Ninhydrin. A Detailed Ab Initio Computational Study. J. Forensic Sci. 2006, 51 (6), 1267–1275.


Page 18 of 30

Page 19 of 30


(14)

Spackman, D. H.; Stein, W. H.; Moore, S. Automatic Recording Apparatus for Use in the Chromatography of Amino Acids. Anal. Chem. 1958, 30 (7), 1190–1206.

(15)

Fimmen, R. L.; Trouts, T. D.; Richter, D. D.; Vasudevan, D. Improved Speciation of Dissolved Organic Nitrogen in Natural Waters: Amide Hydrolysis with Fluorescence Derivatization. J. Environ. Sci. 2008, 20 (10), 1273–1280.

(16)

Friedman, M. Applications of the Ninhydrin Reaction for Analysis of Amino Acids, Peptides, and Proteins to Agricultural and Biomedical Sciences. J. Agric. Food Chem. 2004, 52 (3), 385–406.

(17)

Horstmann, H. J. A Precise Method for the Quantitation of Proteins Taking into Account Their Amino Acid Composition. Anal. Biochem. 1979, 96 (1), 130–138.

(18)

Bogolitsyn, K. G.; Kaplitsin, P. A.; Pochtovalova, A. S. Amino-Acid Composition of Arctic Brown Algae. Chem. Nat. Compd. 2014, 49 (6), 1110–1113.

(19)

Roth, M. Fluorescence Reaction for Amino Acids. Anal. Chem. 1971, 43 (7), 880–882.

(20)

Roth, M. Determination of Ammonia and Organic Amino Compounds. United States Pat. Off. 1972, No. 3,718,432, 1–4.

(21)

Dai, Z.; Wu, Z.; Wang, J.; Wang, X.; Jia, S.; Bazer, F.; Wu, G. Analysis of Polyamines in Biological Samples by HPLC Involving Pre-Column Derivatization with o Phthalaldehyde and N -Acetyl- L -Cysteine. Amino Acids 2014, 46, 1557–1564.

(22)

Molnar-perl, I.; Aniko, V. Stability and Characteristics of the o -Phthaldialdehyde / 3Mercaptopropionic Acid and o -Phthaldialdehyde / N -Acetyl- L -Cysteine Reagents and Their Amino Acid Derivatives Measured By. J. Chromatogr. A 1999, 835, 73–91.

(23)

Schwarz, E. L.; Roberts, W. L.; Pasquali, M. Analysis of Plasma Amino Acids by HPLC with Photodiode Array and Fluorescence Detection. Clin. Chim. Acta 2005, 354 (1–2),



83–90. (24)

Švedas, V. J. K.; Galaev, I. J.; Borisov, I. L.; Berezin, I. V. The Interaction of Amino Acids with O-Phthaldialdehyde: A Kinetic Study and Spectrophotometric Assay of the Reaction Product. Anal. Biochem. 1980, 101 (1), 188–195.

(25)

Wong, O. S.; Sternson, L. A.; Schowen, R. L. Reaction of O-Phthalaldehyde with Alanine and Thiols: Kinetics and Mechanism. J. Am. Chem. Soc. 1985, 107, 6421–6422.

(26)

Vasanits, A.; Kutlán, D.; Sass, P.; Molnár-Perl, I. Retention/Quantitation Properties of the o-Phthaldialdehyde-3-Mercaptopropionic Acid and the o-Phthaldialdehyde-N-Acetyl-LCysteine Amino Acid Derivatives in Reversed-Phase High-Performance Liquid Chromatography. J. Chromatogr. A 2000, 870 (1–2), 271–287.

(27)

Molnár-Perl, I. Derivatization and Chromatographic Behavior of the O-Phthaldialdehyde Amino Acid Derivatives Obtained with Various SH-Group-Containing Additives. J. Chromatogr. A 2001, 913 (1–2), 283–302.

(28)

Hanczkó, R.; Jámbor, A.; Perl, A.; Molnár-Perl, I. Advances in the O-Phthalaldehyde Derivatizations. Comeback to the o-Phthalaldehyde-Ethanethiol Reagent. J. Chromatogr. A 2007, 1163 (1–2), 25–42.

(29)

Godel, H.; Graser, T.; Földi, P.; Pfaender, P.; Fürst, P. Measurement of Free Amino Acids in Human Biological Fluids by High-Performance Liquid Chromatography. J. Chromatogr. A 1984, 297 (C), 49–61.

(30)

Mendez, E.; Gavilanes, J. G. Fluorometric Detection of Peptides after Column Chromatography or on Paper: O -Phthalaldehyde and Fluorescamine. Anal. Biochem. 1976, 72 (1–2), 473–479.

(31)

Rizzo, V.; Anesi, A.; Montalbetti, L.; Bellantoni, G.; Trotti, R.; Melzi d’Eril, G. V.


Page 20 of 30

Page 21 of 30


Reference Values of Neuroactive Amino Acids in the Cerebrospinal Fluid by HighPerformance Liquid Chromatography with Electrochemical and Fluorescence Detection. J. Chromatogr. 1996, 729 (1–2), 181–188. (32)

Molnár-Perl, I. Advancement in the Derivatizations of the Amino Groups with the OPhthaldehyde-Thiol and with the 9-Fluorenylmethyloxycarbonyl Chloride Reagents. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2011, 879 (17–18), 1241–1269.

(33)

Jarret, H.; KD, C.; B, E.; Anderson, J. The Separation of Ophthalaldehyde Derivatives of Amino Acids by Reverse-Phase Chromatrography on Octylsilica Columms. Anal. Biochem. 1986, 153, 189–198.

(34)

Butcher, E. C.; Lowry, O. H. Measurement of Nanogram Quantities of Protein by Hydrolysis Followed by Reaction with Orthophthalaldehyde or Determination of Glutamate. Anal. Biochem. 1976, 76 (2), 502–523.

(35)

Medina Hernández, M. J.; Villanueva Camañas, R. M.; Monfort Cuenca, E.; García Alvarez-Coque, M. C. Determination of the Protein and Free Amino Acid Content in a Sample Using O-Phthalaldehyde and N-Acetyl-L-Cysteine. Analyst 1990, 115 (August), 1125–1128.

(36)

Dong, T.; Knoshaug, E. P.; Davis, R.; Laurens, L. M. L.; Van Wychen, S.; Pienkos, P. T.; Nagle, N. Combined Algal Processing: A Novel Integrated Biorefinery Process to Produce Algal Biofuels and Bioproducts. Algal Res. 2015, 19, 216–323.

(37)

Moore, S.; Stein, W. H. Chromatographic Determination of Amino Acids by the Use of Automatic Recording Equipment. Methods Enzymol. 1963, 6, 819–831.

Figures Figure 1: Reaction mechanism forming the amino acid, O-phthalaldehyde/mercaptopropionic acid (AA/OPA/MPA) analyte




Page 22 of 30

Page 23 of 30


Figure 2: Characteristic absorption spectrum of AA/OPA/MPA derivative. An absorbance peak was observed at 334nm and selected as the analytical wavelength for subsequent analyses in this study.



Figure 3: (A) Absorbance versus concentration plots for bulks amino acid solutions # 1, 2, 3, and 4 (as described in the text). The R2 values for each were 0.9999, 0.9994, 0.9996, and 0.9999, respectively. (B) Error (%) of measured protein concentration versus theoretical calculated concentration.


Page 24 of 30

Page 25 of 30


Figure 4: Matrix interference based on specific process products; OPA derivatization recovery was tested in the presence of 7 compounds typically present in algae-derived process streams, based on a reference sample at 60 µM amino acids



Page 26 of 30

Figure 5: Spectral interference analysis based on absorbance spectra of early stage harvest C. vulgaris (A), S. obliquus (B), N. oculata (C), and D. armatus (D), following acid hydrolysis and OPA/MPA derivatization

A

B

Chlorella vulgaris

2

Scenedesmus obliquus

2

Absorbance (AU)

Diluent OPA/MPA Matrix

1

1

Replicate 1 Replicate 2 Replicate 3

0

0 250

C

300 350 Wavelength (nm)

D

Nannochloropsis oculata

1


400

Desmodesmus armatus

2

Absorbance (AU)

2

250

400

Diluent OPA/MPA Matrix Replicate 1 Replicate 2 Replicate 3

1

0 250


400

0 250



400

Page 27 of 30


Figure 6: Protein measurements of algal biomass following 6N acid digestion, compared to contract laboratory. Species/timepoints are as follows: 1-Chlorella vulgaris (early); 2- Chlorella vulgaris (mid); 3- Chlorella vulgaris (late); 4- Scenedesmus obliquus (early);5- Scenedesmus obliquus (mid); 6-Scenedesmus obliquus (late); 7- Nannochloropsis oculata (early); 8Nannochloropsis oculata (mid);9- Nannochloropsis oculata (late); Desmodesmus armatus (mid), data shown as mean ± stdev of triplicate analyses for all samples

50.0

Weight % Amino Acids

40.0

30.0 Contract Lab Reported Measured AA Content BSA Normalization

20.0

20.3% NPN Normalization

10.0

0.0 1

2

3

4

5

6

7

8

9


10


Page 28 of 30

Tables Table 1: Typical blank-corrected absorbance data for AA/OPA/MPA derivative (Glutamate shown), between 30-55 minutes for 4 different amino acid concentrations (20-101 µM). OPA and MPA concentrations for these experiments were 4mM and 12mM, respectively

20 µM 30 min 35 min 40 min 45 min 50 min 55 min

0.141 0.144 0.146 0.147 0.147 0.147

40 µM

60 µM

80 µM

101 µM

Slope (AU/uM)

Intercept

0.270 0.276 0.279 0.280 0.281 0.282

0.406 0.413 0.416 0.417 0.418 0.418

0.532 0.541 0.545 0.548 0.549 0.55

0.671 0.681 0.687 0.690 0.691 0.692

0.0066 0.0067 0.0067 0.0067 0.0068 0.0068

0.0016 0.004 0.0057 0.0066 0.0073 0.0085


R2 0.9998 0.9998 0.9998 0.9998 0.9999 0.9999

Page 29 of 30


Table 2: Average molar extinction coefficients, absorptivity (cm-1 M-1) calculated for each amino acid, compared to values reported by Svedas et al.21, the ratio is calculated as the values from this study over the previously published absorptivity by Svedas, the relative standard deviation around the ratio was 9.8% Amino Acid

This study (cm-1M-1 )

Svedas21 (cm-1M-1 )

Ratio

Aspartic Acid

6522

4800

1.36

Threonine

5480

5200

1.05

Serine

7149

5500

1.30

Glutamic Acid

6915

4900

1.41

Glycine

7367

5000

1.47

Alanine

7170

5400

1.33

Valine

7115

5600

1.27

Methionine

7108

5200

1.37

Isoleucine

6183

5000

1.24

Leucine

6818

4800

1.42

Tyrosine

5162

5200

0.99

Phenylalanine

6811

5700

1.19

Tryptophan

6404

4700

1.36

Lysine

11462

8800

1.30

Histidine

6250

4800

1.30

Arginine

7235

5700

1.27

Average

6947

5394

1.29



TOC graphic:

Absorbance

2

1

0 250

300

(nm)

350

400


Page 30 of 30

Optimization of Amino Acid Derivatization after ... - ACS Publications

Recommend Documents