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Comparison of heat-induced aggregation of globular proteins Roy J.B.M. Delahaije, Peter A. Wierenga, Marco L.F. Giuseppin, and Harry Gruppen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00927 • Publication Date (Web): 12 May 2015 Downloaded from http://pubs.acs.org on May 17, 2015

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Journal of Agricultural and Food Chemistry

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Comparison of heat-induced aggregation of

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globular proteins

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Roy J.B.M. Delahaije,1 Peter A. Wierenga,1 Marco L.F. Giuseppin,2 and Harry Gruppen1,*

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1

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Wageningen, The Netherlands.

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2

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*Phone: +31 317 483211, E-mail: [email protected]

Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708 WG,

AVEBE, Prins Hendrikplein 20, 9641 GK, Veendam, The Netherlands.

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Abstract

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Typically heat-induced aggregation of proteins is studied using a single protein under various

11

conditions (e.g. temperature). Since different studies use different conditions and methods, a

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mechanistic relation between molecular properties and the aggregation behavior of proteins has

13

not been identified. Therefore, this study investigates the kinetics of heat-induced aggregation

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and size/density of formed aggregates for three different proteins (ovalbumin, β-lactoglobulin

15

and patatin) under various conditions (pH, ionic strength, concentration, temperature). The

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aggregation rate of β-lactoglobulin was slower (>10 times) than that of ovalbumin and patatin.

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Moreover, the conditions (pH, I, C) affected the aggregation kinetics of β-lactoglobulin more

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strongly than for ovalbumin and patatin. In contrast to the kinetics, for all proteins the aggregate

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size/density increased with decreasing electrostatic repulsion. By comparing these proteins under

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these conditions, it became clear that the aggregation behavior cannot easily be correlated to the

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molecular properties (e.g. charge, exposed hydrophobicity).

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ACS Paragon Plus Environment

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Keywords

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β-lactoglobulin, ovalbumin, patatin, pH, ionic strength, concentration

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Introduction

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Heat-induced aggregation of globular proteins is generally described to be caused by the

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exposure of the internal hydrophobic amino acids due to unfolding1, 2. After unfolding and

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exposure of the hydrophobic amino acid residues, the (partially) unfolded protein either refolds

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or irreversibly aggregates with other (partially) unfolded proteins3, 4. The likelihood of refolding

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or aggregation depends on the balance between the increased hydrophobic attraction due to

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unfolding and the mainly electrostatic barrier for aggregation. Qualitatively, aggregation has

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been investigated extensively, mainly in terms of (1) aggregation kinetics5-7, (2) aggregate size8, 9

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and (3) aggregate structure10, 11. These studies typically investigate the effect of conditions (e.g.

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ionic strength) on the aggregation of a single protein (e.g. β-lactoglobulin). A detailed

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mechanistic comparison of the aggregation of different proteins, on the other hand, is still

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missing. Exactly such information may advance the understanding of the aggregation process of

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globular proteins, especially with respect to an overall description of the effect of parameters

39

influencing the aggregation process. Hence, the aim of the present paper is to obtain a

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mechanistic understanding of the aggregation process and determine the effect of the properties

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of three proteins on the aggregation behavior under various conditions.

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The aggregation kinetics (i.e. order (n) and rate (k)) are generally derived from the decrease of

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the concentration of non-aggregated protein in time. Therefore, they provide information on the

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kinetics of protein unfolding and integration of non-aggregated proteins in aggregates, whereas it

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does not contain information on the size and structure of the formed aggregates.


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Aggregation order

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To determine the order of aggregation, two approaches have been described: (A) determination

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of the concentration dependence of the initial aggregation rate5-7 and (B) fitting the decrease of

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the concentration of non-aggregated protein in time with the specific reaction equations. These

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are equations 1 and 2 for 1st and nth order kinetics, respectively12.

Ct = C0 ⋅ e−kt for n = 1 Ct = (C0

1−n

(1) 1

+ (n − 1) ⋅ k ⋅ t ) 1−n for n ≠ 1

(2)

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in which Ct and C0 is the concentration of non-aggregated protein at time t and time 0 [g/L],

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respectively, n is the aggregation order [-] and k is the aggregation rate [(g/L)(1-n)/s].

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These discrepancies between the two approaches are assumed to originate from the fact that the

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aggregation process becomes more complex at longer heating times. This is due to simultaneous

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aggregation of non-aggregated protein with other non-aggregated protein, non-aggregated with

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aggregated proteins, and aggregated proteins with other aggregated proteins.

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For ovalbumin, the initial aggregation rate was shown to be concentration independent. Hence,

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the aggregation process has been described as a first-order reaction6. The aggregation rate of

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β-lactoglobulin, on the other hand, increases with increasing concentration, indicating

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concentration dependence. This resulted in an aggregation order of 1.55,

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aggregation orders of ovalbumin and β-lactoglobulin were shown to be independent of

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temperature6, 14, 15, pH8, 14 and ionic strength6, 16.

7, 13

. In addition, the

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Aggregation rate

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For proteins, the fraction unfolded protein and the diffusion rate of the protein molecules

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typically increase with increasing the temperature17, 18. The fraction unfolded proteins depends


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on the heating temperature relative to the denaturation temperature (Td). For a more fundamental

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comparison of the aggregation properties of different proteins, the proteins are studied at a fixed

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temperature relative to Td, rather than an absolute temperature. This results in a constant fraction

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unfolded protein. An increase of the diffusion rate results in an increased likelihood of the

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proteins to meet, collide and aggregate19, 20. The aggregation rate is also affected by electrostatic

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repulsion. However, a clear link is difficult to make, since the main factors (i.e. ionic strength

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and pH) can have several effects. Increasing the electrostatic repulsion increases both the intra-

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and intermolecular repulsion. The increased intramolecular repulsion decreases the denaturation

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temperature21,

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electrostatic barrier for aggregation23. While the first effect would (when heating at given

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temperature) result in an increase of the aggregation rate, the second would decrease the

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aggregation rate. In addition, a pH change does not only affect the electrostatic interactions, but

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also changes the reactivity of the disulfide bonds. While changes in the electrostatic repulsion

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(i.e. ionic strength and pH) do not affect the aggregation rate of ovalbumin6, 24, the aggregation

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rate of β-lactoglobulin increases with increasing ionic strength (< 0.1 M)16 and pH8,

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difference indicates that the electrostatic repulsion affects both proteins differently. The

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enhancing effect of ionic strength on the aggregation rate of β-lactoglobulin showed that the

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increase of the denaturation temperature was less important than the decrease of the electrostatic

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barrier for aggregation16. At the same time, a higher denaturation temperature and a decrease of

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the reactivity of the disulfide bonds were described to reduce the aggregation rate of

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β-lactoglobulin with decreasing pH14.

22

. The increase of intermolecular repulsion, on the other hand, increases the

14

. This

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Aggregate formation

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To describe the aggregate formation, the size and structure of the aggregates are typically

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determined by scattering techniques (i.e. neutron, x-ray and light scattering). The structure of the

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aggregates is described by the fractal dimension. The fractal dimension was found to be

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independent of the heating temperature15, 25. As temperature only affects the aggregation kinetics

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and not the formation of aggregates, the effect of temperature has been described as a purely

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kinetic effect15. Electrostatic repulsion, however, strongly affects the aggregate formation (size

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as well as structure). A decrease of the electrostatic repulsion, due to an increase of the ionic

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strength and/or a shift of the pH towards the iso-electric point (pI), resulted in the formation of

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larger aggregates of ovalbumin26,

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scattering. At conditions with high electrostatic repulsion (i.e. low ionic strength and pH far from

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the pI), linear aggregates with a fractal dimension of 1.7 were formed by ovalbumin30 and

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β-lactoglobulin31, 32. When the electrostatic repulsion was decreased (i.e. by increasing the ionic

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strength from 10 to 100 mM), the β-lactoglobulin and ovalbumin aggregates become more

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branched. This was indicated by a fractal dimension of 2.015,

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TEM30. When the electrostatic repulsion is minimal (i.e. close to the pI), β-lactoglobulin forms

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even more compact aggregates with a fractal dimension of 3.036. The effect of electrostatic

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repulsion on the aggregate size and structure is explained by the electrostatic barrier for

107

aggregation30, 32, 35. Furthermore, the size of β-lactoglobulin and BSA aggregates increased with

108

increasing concentration9,

109

result of an increase of the ionic strength due to the counter ions of the protein.

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Since β-lactoglobulin has most often been used to study the aggregation process, its aggregation

111

behavior is quite well understood. However, little is known on how this knowledge can be

15, 33, 37

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, β-lactoglobulin8,

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, BSA9 and patatin29, based on light

30, 33-35

, and confirmed by cryo-

. This concentration dependence has been postulated to be a


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extrapolated to describe the aggregation of other proteins. To obtain a more mechanistic

113

understanding of the effect of protein molecular properties on the aggregation of globular

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proteins, this study compares the aggregation process of different proteins. Hence, the

115

differences in the kinetics and factors influencing the aggregation of three proteins

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(β-lactoglobulin, ovalbumin and patatin) under various conditions (pH, ionic strength,

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temperature and protein concentration) are studied. The three proteins are selected as their

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similarities (globular structure, pI and molecular weight) and differences (surface charge,

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exposed hydrophobicity and number of disulfide bridges) (table 1) enable a comparison of the

120

effect of charge, hydrophobicity and disulfide bridges on the aggregation behavior.

121 122

Materials and methods

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Materials

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Ovalbumin (A-5503, Lot nᵒ 031M7008V; pI = 5.19; protein content 92 % (N x 6.22)38 of which

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≥ 98 % ovalbumin (based on agarose gel electrophorese)) and β-lactoglobulin (L-0130, Lot nᵒ

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SLBC2933V; pI = 4.83; protein content of 94 % (N x 6.38)38 of which 99 % β-lactoglobulin

127

(based on PAGE)) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Potatoes were

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provided by AVEBE BA (Veendam, The Netherlands). Patatin was isolated from potato juice as

129

described previously39, except that gel filtration was performed on a Superdex 200 column (52 x

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10 cm). The obtained patatin fraction (pI = 5.25; purity ≥ 90 % based on analytical

131

size-exclusion chromatography) was dialyzed against demineralized water, freeze-dried and

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stored at -20 °C. All other chemicals were of analytical grade and purchased from either

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Sigma-Aldrich or Merck.

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Differential scanning calorimetry (DSC)

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The denaturation temperature of the proteins was determined using a VP-DSC MicroCalorimeter

137

(MicroCal Inc., Northampton, MA, USA). The proteins were dissolved in 10 mM sodium

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phosphate buffer pH 7.0 at a concentration of 2 g/L. Subsequently, thermograms were recorded

139

from 20 to 100 °C at a heating rate of 1 °C/min. The denaturation temperatures (Td) of

140

ovalbumin, β-lactoglobulin and patatin were found to be 77.5, 75.0 and 60.0 °C, respectively

141

(supporting information). This is in close agreement with the denaturation temperatures reported

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in literature6, 21, 39.

143 144

Heat-induced aggregation

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Heat-induced aggregation was performed in a water bath. Aliquots of 1 mL were heated for

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different time intervals (0, 1, 2, 5, 10, 20, 30, 40, 50, 60, 100, 200, 300, 400, 500, 1500, 1800,

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3000, 4500 and 10080 min) in Kimax tubes. After heating, the samples were cooled on ice-

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water. Four different sets of experiments were performed:

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Effect of temperature

150

Proteins were dissolved in 10 mM sodium phosphate buffer pH 7.0 at a concentration of 2 g/L.

151

Subsequently, these solutions were heated at temperatures at the same distance from the

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denaturation temperature (Td): Td - 10 °C, Td - 5 °C, Td and Td + 5 °C. This was chosen to ensure

153

as well as possible a similar rate of unfolding for all proteins. The reason is that at Td, for all

154

proteins, the native and unfolded proteins are present in equal amounts12, 17. This equilibrium

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between native and unfolded protein shifts towards the native protein with decreasing and

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towards unfolded protein with increasing temperature. This equilibrium is, at a certain

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temperature from Td (e.g. Td - 5 °C), assumed to be equal for all proteins. For the incubations at


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the two highest temperatures, additional time intervals (i.e. 0.5, 1.5, 2.5, 3, 4, 7.5, 12.5, 15, 17.5

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and 25 min) were included.

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The aggregation rate was determined based on the decrease of the concentration of

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non-aggregated protein in time using equation 1 or 2. From the temperature dependence of the

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aggregation rate (k), the activation energy (Ea) was calculated by the Arrhenius equation.

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Effect of pH

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Proteins were dissolved in 10 mM sodium phosphate buffer pH 7.0, pH 6.0 and pH 5.0 at a

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concentration of 2 g/L. Subsequently, the solutions were heated at Td - 5 °C.

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Effect of ionic strength

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Proteins were dissolved in 10 mM sodium phosphate buffer pH 7.0 in the presence of absence of

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10, 40 and 90 mM NaCl at a concentration of 2 g/L. Subsequently, the solutions were heated at

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Td - 5 °C.

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Effect of protein concentration

171

Proteins were dissolved in 10 mM sodium phosphate buffer pH 7.0 at concentrations of 1, 2, 5

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and 10 g/L. Subsequently, the solutions were heated at Td - 5 °C.

173 174

Size-exclusion chromatography (SEC)

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The concentration of non-aggregated protein (i.e. monomer concentration for ovalbumin and

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dimer concentration for β-lactoglobulin and patatin) was determined using SEC on an Äkta

177

Micro equipped with a Superdex 200 PC 3.2/30 column (GE Healthcare, Uppsala, Sweden).

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Prior to analysis, the samples were centrifuged (16,100g, 10 min, 20 °C) to remove insoluble

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aggregates. Subsequently, samples (20 µL) were injected and eluted with 10 mM sodium

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phosphate buffer pH 7.0 at a flow rate of 0.06 mL/min. The elution was monitored at 214 nm.


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The calibration was performed with globular proteins with a mass range of 13.7 - 67 kDa (GE

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Healthcare). The typical experimental error for the determination of the amount of non-

183

aggregated protein was < 10 % as determined from duplicate time incubations of ovalbumin and

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patatin, 2 g/L, Td - 5 °C, 10 mM sodium phosphate buffer pH 7.0.

185 186

Determination of the aggregation kinetics

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The aggregation kinetics are determined in several ways: (1) concentration dependence of the

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time required to aggregate half of the protein (i.e. th as indication of the aggregation rate), (2)

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fitting the decrease of the concentration of non-aggregated protein in time using equations 1 and

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2 and (3) fitting the natural logarithm of concentration (n = 1.0), reciprocal of the square root of

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concentration (n = 1.5) and the reciprocal of the concentration (n= 2.0) against time with a linear

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function, fixing the intercept with the y-axis corresponding to the initial concentration (C0). The

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first approach only includes the data points Ct ≥ 0.5C0, whereas the other two approaches are

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applied to the regime Ct ≥ 0.05C0. For the second approach the data is fitted by minimizing the

195

sum of squares error using the Microsoft Excel solver.

196 197

Static light scattering (SLS)

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The scattered light intensity was determined in time using a Malvern Zetasizer Nano ZS

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(Malvern Instruments, Worcestershire, UK). The protein solutions (as described in the section

200

heat-induced aggregation) were filtered over a 0.1 µm PTFE filter (Puradisc 13; Whatman, Kent,

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UK). Subsequently, the filtered solutions were heated at the temperatures described in the section

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on heat-induced aggregation. The scattered light intensity was measured every 30 seconds for

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7200 seconds. The intensity of the scattered light is generally described by equation 313, 40.


9


I (q ) ∝ K ⋅ C ⋅ M w ⋅ P (q) ⋅ S (q )

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(3)

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in which I(q) is the intensity of the scattered light at angle q (i.e. 173°), K is an optical constant,

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c is the concentration [g/L], Mw is the molar mass of the particles [g/mol], P(q) is the particle

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form factor which describes the shape and size of the particles and S(q) is the structure factor

207

that describes the spatial arrangement of the particles.

208

As the concentration (C) and the optical constant (K) are constant during and between the

209

measurements equation 3 can be simplified to:

I ( q) ∝ M w ⋅ P( q ) ⋅ S ( q)

(4)

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This shows that an increase of the scattered light intensity relates to an increase of the aggregate

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size and/or a more dense aggregate structure.

212 213

Results and discussion

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Aggregation order

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Generally, the aggregation kinetics are described by the aggregation order and aggregation rate.

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The first parameter used to compare the aggregation behavior of different proteins is the

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aggregation order (n). To exclude the effect of aggregation rate and thereby only obtain

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information on the influence of conditions on the aggregation order, the decrease of the

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concentration of non-aggregated protein is plotted against the time divided by the time required

220

to aggregate half of the proteins (th) (figures 1A-C). All curves of one protein superimpose onto

221

one master curve, indicating that the aggregation order is not affected by temperature,

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concentration, pH and ionic strength. In other words, the general aggregation process of each

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protein can be described by a single aggregation order under the different conditions studied.

224

The small variations for ovalbumin and patatin at Td and Td + 5°C were ascribed to small


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inaccuracies as a result of the fast aggregation rate (i.e. indicated by a th ≤ 2 min; table 3). The

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observed discrepancies for patatin under conditions with a slower aggregation rates (i.e.

227

indicated by a th ≥ 50 min; table 3) are not completely understood, but could be caused by a

228

stronger tendency to form larger aggregates in the initial stage of the aggregation process.

229

The aggregation order for each of the three proteins was determined using three approaches. The

230

first approach derives the aggregation order from the concentration dependence of th (figure 2

231

and table 2). The th is concentration independent for ovalbumin and patatin, indicating a first

232

order reaction. For ovalbumin, this is in line with previous data6, whereas for patatin no previous

233

data is present. For β-lactoglobulin, on the other hand, th depends on the concentration. The slope

234

of the concentration dependence of th is -0.5. This shows that, within the experimental error, th

235

scales with √C, implying an order of 1.57. This is in line with literature5, 7. The second approach

236

is based on fitting the decrease of the concentration of non-aggregated protein in time using

237

equations 1 and 2. For all proteins, the order of the aggregation process is higher than second

238

order kinetics (i.e. n = 2.5, 2.1 and 2.9 for β-lactoglobulin, ovalbumin and patatin, respectively)

239

(table 2). This indicates a complex aggregation process, in which non-aggregated proteins, for

240

example, aggregate with other non-aggregated proteins as well as aggregated proteins. The third

241

approach is based on fitting the decrease of ln Ct, 1/√Ct or 1/Ct in time to a linear function. This

242

resulted in the best fit (i.e. R2) of an aggregation order of 2.0 for ovalbumin and patatin (R2 =

243

0.95 ± 0.04 and 0.97 ± 0.01 for ovalbumin and patatin, respectively) (table 2). Moreover, it

244

showed that the first order kinetics for ovalbumin and patatin, as determined by the first

245

approach, the low R2 (i.e. R2 = 0.28) disqualifies the first order kinetics. This discrepancy

246

between the concentration and time fit has been described previously41. Since the time fit

247

approaches include all the data for fitting, it is concluded that the overall aggregation can be well


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248

described based on second order kinetics. The best for β-lactoglobulin was an aggregation order

249

of 1.5 (R2 = 0.96 ± 0.04). The fit of the data of β-lactoglobulin for a second order reaction is,

250

however, not significantly different from that of an order of 1.5. Although the aggregation

251

process of β-lactoglobulin can overall be described by an aggregation order of 1.5 or 2.0, the

252

aggregation process at pH 5.0 shows a better fit with first order kinetics (i.e. R2 = 0.99 and 0.84

253

for n = 1.0 and 1.5, respectively).

254

Summarizing, the aggregation process of all tested proteins, β-lactoglobulin, ovalbumin and

255

patatin, is described by an order of 2.0 (R2 > 0.90 for all proteins). The aggregation process of

256

β-lactoglobulin at pH 5.0 is a first order process.

257 258

Aggregation rate

259

In the previous section, the reaction order was determined from rescaled data (i.e. t/th). The

260

aggregation rate (k) is determined by fitting the decrease of the concentration of non-aggregated

261

protein versus real time (t) with equation 2. To allow quantitative comparison of the aggregation

262

rates, the data of all proteins is fitted with an aggregation order of 2.0 (R2 > 0.90 for all proteins)

263

(table 2).

264

Effect of temperature (2 g/L in 10 mM buffer pH 7.0)

265

Quantitatively, the effect of temperature on the aggregation rate varies significantly between the

266

proteins (figures 1D-F and table 3). Whereas the decrease of the concentration of non-aggregated

267

protein for ovalbumin and patatin at Td - 5°C (pH = 7.0, I = 10 mM and C = 2 g/L) is in the same

268

order of magnitude (i.e. th ~ 10 min), the decrease of non-aggregated β-lactoglobulin is

269

significantly slower (i.e. th ~ 220 min) (table 3). In addition, the temperature dependence of the

270

aggregation rate varies significantly between the proteins. This temperature dependence is


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271

reflected in the activation energy (Ea), calculated using the Arrhenius equation, which was found

272

to be 343, 293 and 220 kJ/mol for patatin, ovalbumin and β-lactoglobulin, respectively

273

(supporting information). These activation energies are lower than reported in literature (i.e. ~

274

260-300 kJ/mol for β-lactoglobulin42,

275

conditions (i.e. pH 6.5-7.0).

276

Summarizing, the aggregation rate decreases from ovalbumin ≥ patatin >> β-lactoglobulin.

277

Based on the relative exposed hydrophobicity, the order was expected to be β-lactoglobulin >

278

patatin > ovalbumin. Based on the net surface charge density no large differences were expected.

279

Considering the possibility that the free sulfhydryl groups can interchange with disulfide

280

bonds44, the expected order is ovalbumin > β-lactoglobulin > patatin. From the previous it is

281

concluded that there is no clear unambiguous relation between one of these protein properties

282

and the determined rate of aggregation. One parameter that has not often been considered is the

283

extent of protein unfolding. The extent of unfolding may be limited by the presence of disulfide

284

bonds. Therefore, patatin and ovalbumin are expected unfold more completely than β-

285

lactoglobulin, due to the presence of 2 disulfide bonds in β-lactoglobulin. Consequently, less

286

hydrophobic amino acids become exposed for β-lactoglobulin than for the other two proteins,

287

leading to less non-covalent interactions.

288

Effect of pH (2 g/L in 10 mM buffer heated at Td - 5 °C)

289

The aggregation rate of ovalbumin is not affected by the pH in the range from pH 5 to pH 7

290

(figure 1D and table 3). The aggregation rates of β-lactoglobulin and patatin, on the other hand,

291

are slower at a pH closer to the iso-electric point (pI), with th of 223 and 10.8 min at pH 7.0,

292

compared to th of 1052 and 73 min at pH 5.0 for β-lactoglobulin and patatin, respectively (figures

293

1E and F and table 3).

43

and 338 kJ/mol for ovalbumin24) under similar


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Page 14 of 32

294

For patatin, the effect of pH seems to be due to a difference in the denaturation temperature.

295

When the aggregation rates of patatin at pH 5 and pH 6 are compared to pH 7 at different

296

temperatures, they are similar to pH 7 at a temperature between Td - 5 °C and Td - 10 °C (table

297

3). This corresponds with a theoretical increase of Td of ~ 4 °C, which is in close agreement with

298

literature, i.e. 3 °C increase of Td from pH 7 to pH 645. For β-lactoglobulin, the aggregation rate

299

at pH 5 and pH 6 is slower than at pH 7 and Td - 10 °C (table 3). The observed decrease of the

300

aggregation rate by shifting the pH towards the pI is in line with literature5,

301

based on the temperature dependence of the aggregation rate (table 3), the Td should increase by

302

7-10 °C to explain the decrease in aggregation rate. Experimentally, the Td of β-lactoglobulin

303

was only reported to increase by 2 °C when the pH was decreased from pH 7 to pH 516.

304

Consequently, another factor also has to be influenced by the pH change. The association state of

305

β-lactoglobulin, as measured by size-exclusion chromatography, was not affected by the pH

306

changes (data not shown). The thiol groups, on the other hand, become less reactive with

307

decreasing pH8. Therefore, the decreased reactivity of the thiol groups can explain the observed

308

differences.

309

Effect of ionic strength (2 g/L in buffer pH 7.0 heated at Td - 5 °C) and concentration (10 mM

310

buffer pH 7.0 heated at Td - 5 °C)

311

For β-lactoglobulin, the aggregation rate increases with increasing ionic strength (i.e. th decreases

312

from 223 min at 10 mM to 87 min at 100 mM) and protein concentration (i.e. th decreases from

313

255 min at 1 g/L to 84 min at 10 g/L) (figure 1E and table 3). The effect of ionic strength is

314

explained by a decrease of the electrostatic repulsion within and between the protein molecules.

315

This was described to result in an increase of the denaturation temperature (i.e. 2 and 5 °C from 0

316

to 100 mM for β-lactoglobulin and BSA, respectively)21,

22

7, 8, 14

. However,

. In addition, it also results in a


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317

decrease of the electrostatic barrier for aggregation23. As in this case the aggregation rate

318

increases rather than decreases, the enhancing effect of electrostatics was more important than

319

the observed increase of the denaturation temperature. The effect of concentration on the

320

aggregation rate is line with previous data7. The effect is postulated to be caused by the fact that

321

at higher protein concentrations the absolute number of unfolded protein increases, resulting in

322

an increased likelihood of proteins to meet, collide and aggregate. In contrast to the observation

323

for β-lactoglobulin, the aggregation rates of ovalbumin and patatin are neither affected by the

324

ionic strength nor by the protein concentration (figures 1D and F). These results show that it is

325

important to use different proteins, as the behavior of proteins cannot be extrapolated from one

326

protein to another.

327 328

Formation of aggregates

329

Whereas some protein solutions remained transparent after heating, others became translucent or

330

opaque. These clear visual differences between the heated protein solutions are also reflected in

331

the ionic strength dependence of the aggregated protein in time determined by size-exclusion

332

chromatography (SEC) (figure 3).

333

At all ionic strengths, the UV peak area corresponding to the aggregates initially increases and

334

subsequently decreases. This decrease shifted to shorter heating times with increasing ionic

335

strength. This decrease at increased ionic strength and longer times is due to the formation of

336

larger aggregates, which are removed during the centrifugation step prior to SEC analysis.

337

To obtain insights in the effect of the conditions (e.g. pH) on the formation of aggregates, the

338

scattered light intensity is monitored in time (figures 4A-C). According to equation 4, an increase

339

in the scattered light intensity is caused by an increase of the aggregate size and/or alterations of


15


Page 16 of 32

340

the aggregate shape (at a given concentration). Based on the aggregation kinetics (table 3), the

341

time is recalculated into the fraction aggregated protein (figures 4D-F). This provides insights in

342

the aggregation mechanism (e.g. preference towards the formation of small or large aggregates).

343

For ovalbumin and β-lactoglobulin, the scattered light intensity does not significantly increase

344

when the proteins are heated at Td - 5 °C in a concentration of 2 g/L. For patatin, on the other

345

hand, the intensity of the scattered light increases significantly (i.e. I/I0 = 8 after 7200 seconds).

346

This indicates that the patatin aggregates are larger and/or denser than the ovalbumin and

347

β-lactoglobulin aggregates. These differences cannot be explained based on the aggregation

348

kinetics (i.e. th of ovalbumin and patatin is similar). This shows that for patatin the formation of

349

less, but larger aggregates is favored over more, smaller aggregates as is the case for ovalbumin

350

and β-lactoglobulin under these conditions.

351

Effect of pH and ionic strength

352

For β-lactoglobulin, a lag phase precedes the intensity increase (figure 4B). Moreover, the

353

intensity of the scattered light in time increases for all three proteins (i.e. ovalbumin,

354

β-lactoglobulin and patatin) with increasing ionic strength or a shift of the pH towards the pI

355

(figures 4A-C). The intensity, however, increases more strongly for patatin (i.e. I/I0 = 47 and 12

356

after 200 seconds for pH 5 and 100 mM, respectively) compared to β-lactoglobulin (i.e. I/I0 = 10

357

and 22 after 7200 seconds for pH 5 and 100 mM, respectively) and ovalbumin (i.e. I/I0 = 12 after

358

900 seconds and 8 after 7200 seconds for pH 5 and 100 mM, respectively). The observed lag

359

phase for β-lactoglobulin has also been reported in literature where it is ascribed to the

360

monomer-dimer equilibrium46 or the formation of small aggregates16. Furthermore, it was

361

concluded that a decrease of the electrostatic repulsion results in the formation of larger and/or

362

denser aggregates. This is explained by a lower electrostatic repulsion between the aggregates.


16

Page 17 of 32


363

Consequently, aggregates can continue to grow to larger sizes and/or the formed aggregates are

364

denser. In literature, the molecular mass and size of β-lactoglobulin aggregates were also

365

described to increase when the pH shifts towards the pI and/or the ionic strength increases8, 47, 48.

366

However, as shown, this effect is not the same for all proteins. The difference among the proteins

367

indicates that the magnitude by which the aggregate size/structure in time is affected by

368

electrostatics depends on the protein.

369

Recalculating time into the fraction aggregated protein provides information on the mechanism

370

of aggregation (figures 4D-F). Close to the pI (i.e. pH 5), for patatin and β-lactoglobulin, the

371

intensity increases with the incorporation of even a minor fraction of the protein into the

372

aggregates (i.e. ~ 5 %) (figures 4E and F). For ovalbumin, the intensity increases after ~ 25 % of

373

the protein aggregated (figure 4D). At a pH away from the pI or at lower ionic strength, a larger

374

fraction aggregated protein is required for the intensity to increase. These results show that for

375

patatin and β-lactoglobulin at low electrostatic repulsion (i.e. pH 5) even in the initial stages (i.e.

376

< 10 % aggregated protein) large and/or dense aggregates are formed. For ovalbumin, on the

377

other hand, the lag phase indicates that initially (i.e. < 25 % aggregated protein) smaller and/or

378

more open aggregates are formed. Subsequently, at a higher fraction of aggregated protein, large

379

and/or denser aggregates are formed. Similarly, also at higher electrostatic repulsion initially

380

smaller and/or more open aggregates are formed, followed by the formation of larger and/or

381

denser aggregates. The differences between the proteins show that patatin has the highest

382

tendency to form larger and/or denser aggregates.

383

Effect of concentration and temperature

384

In general, changes in temperature or concentration only resulted in minor changes of the light

385

scattering intensity as function of heating time (figures 4A-C). This shows that these two


17


Page 18 of 32

386

parameters do not affect the aggregate formation (i.e. size and structure) as strongly as ionic

387

strength or pH. For patatin, the intensity increase upon heating is concentration independent,

388

whereas it decreases with decreasing temperature (i.e. I/I0 = 4 after 7200 seconds) (figure 4C).

389

For β-lactoglobulin and ovalbumin, the intensity in time increases slightly with increasing

390

concentration (i.e. I/I0 = 4 and 3 at 10 g/L after 7200 seconds for β-lactoglobulin and ovalbumin,

391

respectively) (figures 4A and B). This increase is in line with literature, as the mass and size of

392

β-lactoglobulin aggregates have been described to increase with concentration (at higher

393

concentrations until 110 g/L)7, 28, 49. The effect of temperature on the aggregation of patatin is

394

expected to be purely determined by kinetics, as a consequence the aggregate formation in time

395

is reduced.

396

From the intensity as function of the fraction aggregated protein it can be observed that, even at

397

large fractions of aggregated protein, the intensity is not affected by concentration and

398

temperature. This confirms that, also after correcting for the aggregation kinetics, the aggregate

399

size and structure are, within the tested temperature and concentration range, independent of

400

temperature and concentration. Hence, at high electrostatic repulsion (i.e. pH 7.0 and 10 mM),

401

relatively small and/or open aggregates are formed.

402

In summary, aggregate formation is limited by the electrostatic repulsion between proteins or

403

protein aggregates due to their charge. In case of charge screening (i.e. increased ionic strength)

404

or a decreased surface charge (i.e. pH closer to pI), this electrostatic barrier decreases and larger

405

and/or denser aggregates are formed. Concentration and temperature, on the other hand, did not

406

strongly affect the aggregate size and/or structure. Although for all proteins aggregate formation

407

was qualitatively similar, quantitatively the behavior was significantly different. This shows that

408

the information on one protein cannot directly be related to another protein. In general,


18

Page 19 of 32


409

ovalbumin showed to have the least tendency to form larger and/or denser aggregates, followed

410

by β-lactoglobulin. Patatin, on the other hand, directly forms large and/or dense aggregates.

411

In conclusion, the comparison of different proteins shows that the sensitivity towards system

412

conditions (i.e. pH, ionic strength and concentration) varies significantly between the proteins.

413

While it was expected that the aggregation behavior relates to the molecular properties of a

414

protein, there is no clear link between the observed aggregation behavior and known protein

415

molecular properties such as surface charge and exposed hydrophobicity. This was for instance

416

illustrated by the lower tendency of ovalbumin to form larger, denser aggregates than patatin and

417

β-lactoglobulin, while the surface charge of these proteins is similar. Therefore, the generic

418

relations commonly assumed in literature need to be considered with care.

419

In addition, although the classical reaction rate kinetics are often used to describe the aggregation

420

process of proteins it was shown to contain little information about the actual size/density of the

421

formed aggregates. Consequently, for a more complete description of the aggregation process,

422

not only the loss of monomers, but also a detailed analysis of the aggregates is required.

423

For a fundamental understanding of the influence of molecular properties on the aggregation

424

process and to exclude factors relating to differences in unfolding behavior, it is important to

425

compare the aggregation behavior at a constant distance from Td. This is in contrast to the fact

426

that for practical situations a comparison at a constant temperature is more valuable (i.e.

427

realistic).

428 429

Notes

430

The authors declare no competing financial interest.

431


19


Page 20 of 32

432

Supporting information

433

Supporting information available:

434

Thermograms of the heat-induced unfolding of ovalbumin, β-lactoglobulin and patatin (2 g/L, 10

435

mM sodium phosphate buffer pH 7.0).

436

Arrhenius plots of ovalbumin, β-lactoglobulin and patatin (2 g/L, 10 mM sodium phosphate

437

buffer pH 7.0).

438 439

References

440

1.

441

176.

442

2.

443

USA, 2002.

444

3.

Lumry, R.; Eyring, H., Conformation changes of proteins. J. Phys. Chem. 1954, 58, 110-120.

445

4.

Mahler, H.-C.; Müller, R.; Frieβ, W.; Delille, A.; Matheus, S., Induction and analysis of aggregates in a

446

liquid IgG1-antibody formulation. Eur. J. Pharm. Biopharm. 2005, 59, 407-417.

447

5.

448

Biochem. 1994, 226, 883-889.

449

6.

450

aggregation of ovalbumin at neutral pH described by irreversible first-order kinetics. Protein Sci. 2003, 12, 2693-

451

2703.

452

7.

453

induced denaturation. Macromolecules 1999, 32, 6120-6127.

454

8.

455

J. Agric. Food Chem. 1999, 47, 1898-1905.

456

9.

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of bovine serum albumin studied by asymmetrical flow field-flow fractionation. Anal. Chim. Acta 2010, 675, 191-

458

198.

Pauling, L., Protein interactions: aggregation of globular proteins. Discuss. Faraday Soc. 1953, 13, 170-

Creighton, T. E., Proteins: structure and molecular properties. 2 ed.; W.H. Freeman: New York, NY,

Roefs, S. P. F. M.; de Kruif, K. G., A model for the denaturation and aggregation of β-lactoglobulin. Eur. J.

Weijers, M.; Barneveld, P. A.; Cohen Stuart, M. A.; Visschers, R. W., Heat-induced denaturation and

Le Bon, C.; Nicolai, T.; Durand, D., Kinetics of aggregation and gelation of globular proteins after heat-

Hoffmann, M. A. M.; van Mil, P. J. J. M., Heat-induced aggregation of β-lactoglobulin as a function of pH.

Yohannes, G.; Wiedmer, S. K.; Elomaa, M.; Jussila, M.; Aseyev, V.; Riekkola, M.-L., Thermal aggregation


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10.

460

globular protein gels at pH 7. Ovalbumin. Macromolecules 2004, 37, 8709-8714.

461

11.

462

aggregation? Biophys. J. 2007, 92, 1336-1342.

463

12.

van Boekel, M. A. J. S., Kinetic modeling of reactions in foods. CRC Press: Boca Raton, FL, USA, 2009.

464

13.

Hoffmann, M. A. M.; Roefs, S. P. F. M.; Verheul, M.; van Mil, P. J. J. M.; de Kruif, K. G., Aggregation of

465

β-lactoglobulin studied by in situ light scattering. J. Dairy Res. 1996, 63, 423-440.

466

14.

467

pH, heating temperature, and protein composition. J. Agric. Food Chem. 1998, 46, 4909-4916.

468

15.

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Int. J. Food Sci. Technol. 1999, 34, 451-465.

470

16.

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Agric. Food Chem. 1998, 46, 896-903.

472

17.

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analysis. J. Pharm. Sci. 2009, 98, 2909-2934.

474

18.

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2010, 390, 89-99.

476

19.

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solution: mechanism and driving forces in nonnative protein aggregation. Pharm. Res. 2003, 20, 1325-1336.

478

20.

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Biotechnol. Bioeng. 1997, 54, 333-343.

480

21.

481

transitions during heat denaturation as studied by differential scanning calorimetry. Food Hydrocolloids 2009, 23,

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2287-2293.

483

22.

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denaturation. Effects of ionic strength and SDS concentration. Int. J. Biol. Macromol. 1997, 20, 193-204.

485

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486

620.

Weijers, M.; Visschers, R. W.; Nicolai, T., Influence of the ionic strength on the structure of heat-set

Krebs, M. R. H.; Devlin, G. L.; Donald, A. M., Protein particulates: another generic form of protein

Verheul, M.; Roefs, S. P. F. M., Structure of particulate whey protein gels: effect of NaCl, concentration,

Le Bon, C.; Nicolai, T.; Durand, D., Growth and structure of aggregates of heat-denatured β-lactoglobulin.

Verheul, M.; Roefs, S. P. F. M.; de Kruif, K. G., Kinetics of heat-induced aggregation of β-lactoglobulin. J.

Mahler, H.-C.; Friess, W.; Grauschopf, U.; Kiese, S., Protein aggregation: pathways, induction factors and

Wang, W.; Nema, S.; Teagarden, D., Protein aggregation - pathways and influencing factors. Int. J. Pharm.

Chi, E. Y.; Krishnan, S.; Randolph, T. W.; Carpenter, J. F., Physical stability of proteins in aqueous

Speed, M. A.; King, J.; Wang, D. I. C., Polymerization mechanism of polypeptide chain aggregation.

Haug, I. J.; Skar, H. M.; Vegarud, G. E.; Langsrud, T.; Draget, K. I., Electrostatic effects on β-lactoglobulin

Giancola, C.; De Sena, C.; Fessas, D.; Graziano, G.; Barone, G., DSC studies on bovine serum albumin

De Young, L. R.; Fink, A. L.; Dill, K. A., Aggregation of globular proteins. Acc. Chem. Res. 1993, 26, 614-


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24.

488

generation of aggregation-prone conformers. Biomacromolecules 2007, 8, 1648-1656.

489

25.

490

shear modulus of heat-set globular protein gels. Soft Matter 2008, 4, 893-900.

491

26.

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property. J. Agric. Food Chem. 1996, 44, 2086-2090.

493

27.

494

denatured ovalbumin investigated with a multiangle laser light scattering technique. J. Food Sci. 2008, 73, C41-C49.

495

28.

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globular proteins. Phys. A 2002, 304, 253-265.

497

29.

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patatin studied in situ. J. Agric. Food Chem. 1999, 47, 4600-4605.

499

30.

500

of ovalbumin. Macromolecules 2002, 35, 4753-4762.

501

31.

502

Int. J. Biol. Macromol. 2008, 43, 129-135.

503

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504

of aggregates and gels formed by heat-denatured whey protein isolate and β-lactoglobulin at neutral pH. J. Agric.

505

Food Chem. 2007, 55, 3104-3111.

506

33.

507

induced aggregation of the globular protein β-lactoglobulin at pH 7. Int. J. Biol. Macromol. 2004, 34, 21-28.

508

34.

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denaturation of globular proteins. Macromolecules 1994, 27, 583-589.

510

35.

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protein gels at pH 7. β-lactoglobulin. Macromolecules 2004, 37, 8703-8708.

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36.

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disaggregation of native β-lactoglobulin in low salt. Langmuir 2013, 29, 4584-4593.

Broersen, K.; Weijers, M.; de Groot, J.; Hamer, R. J.; de Jongh, H. H. J., Effect of protein charge on the

Mehalebi, S.; Nicolai, T.; Durand, D., The influence of electrostatic interaction on the structure and the

Mine, Y., Laser light scattering study on the heat-induced ovalbumin aggregates related to its gelling

Choi, S. J.; Moon, T. W., Influence of sodium chloride and glucose on the aggregation behavior of heat-

Durand, D.; Gimel, J. C.; Nicolai, T., Aggregation, gelation and phase separation of heat denatured

Pots, A. M.; ten Grotenhuis, E.; Gruppen, H.; Voragen, A. G. J.; de Kruif, K. G., Thermal aggregation of

Weijers, M.; Visschers, R. W.; Nicolai, T., Light scattering study of heat-induced aggregation and gelation

Mehalebi, S.; Nicolai, T.; Durand, D., Light scattering study of heat-denatured globular protein aggregates.

Mahmoudi, N.; Mehalebi, S.; Nicolai, T.; Durand, D.; Riaublanc, A., Light-scattering study of the structure

Baussay, K.; Le Bon, C.; Nicolai, T.; Durand, D.; Busnel, J. P., Influence of the ionic strength on the heat-

Gimel, J. C.; Durand, D.; Nicolai, T., Structure and distribution of aggregates formed after heat-induced

Pouzot, M.; Durand, D.; Nicolai, T., Influence of the ionic strength on the structure of heat-set globular

Yan, Y.; Seeman, D.; Zheng, B.; Kizilay, E.; Xu, Y.; Dubin, P. L., pH-dependent aggregation and


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37.

515

β-lactoglobulin aggregates. J. Agric. Food Chem. 1997, 45, 2949-2957.

516

38.

517

patatin gels compared with β-lactoglobulin, ovalbumin, and glycinin. J. Sci. Food Agric. 2011, 91, 253-261.

518

39.

519

P.; Voragen, A. G. J., Effects of pH and heat treatments on the structure and solubility of potato proteins in different

520

preparations. J. Agric. Food Chem. 2001, 49, 4889-4897.

521

40.

522

fractionation. John Wiley: Hoboken, NJ, USA, 2011.

523

41.

524

1996, 61, 477-486.

525

42.

526

reconstituted whole milk. J. Agric. Food Chem. 1996, 44, 422-428.

527

43.

528

1988, 53, 258-263.

529

44.

530

reactions on the heat-induced aggregation kinetics of β-lactoglobulin. Int. Dairy J. 2007, 17, 1034-1042.

531

45.

532

structural stability of patatin. J. Agric. Food Chem. 1998, 46, 2546-2553.

533

46.

534

dynamic light scattering. Int. Dairy J. 1996, 6, 343-357.

535

47.

536

lactoglobulin heat aggregation at high pH. Biophys. J. 2000, 79, 1030-1038.

537

48.

538

characterization of thermally-induced β-lactoglobulin aggregates. J. Food Sci. 2010, 75, E261-E268.

539

49.

540

kinetics. Biomacromolecules 2010, 11, 864-871.

Hoffmann, M. A. M.; Sala, G.; Olieman, C.; de Kruif, K. G., Molecular mass distributions of heat-induced

Creusot, N.; Wierenga, P. A.; Laus, M. C.; Giuseppin, M. L. F.; Gruppen, H., Rheological properties of

van Koningsveld, G. A.; Gruppen, H.; de Jongh, H. H. J.; Wijngaards, G.; van Boekel, M. A. J. S.; Walstra,

Podzimek, S., Light scattering, size exclusion chromatography and asymmetric flow field flow

Van Boekel, M. A. J. S., Statistical aspects of kinetic modeling for food science problems. J. Food Sci.

Anema, S. G.; McKenna, A. B., Reaction kinetics of thermal denaturation of whey proteins in heated

Dannenberg, F.; Kessler, H.-G., Reaction kinetics of the denaturation of whey proteins in milk. J. Food Sci.

Mounsey, J. S.; O’Kennedy, B. T., Conditions limiting the influence of thiol–disulphide interchange

Pots, A. M.; de Jongh, H. H. J.; Gruppen, H.; Hessing, M.; Voragen, A. G. J., The pH dependence of the

Elofsson, U. M.; Dejmek, P.; Paulsson, M. A., Heat-induced aggregation of β-lactoglobulin studied by

Bauer, R.; Carrotta, R.; Rischel, C.; Øgendal, L., Characterization and isolation of intermediates in β-

Zúñiga, R.; Tolkach, A.; Kulozik, U.; Aguilera, J., Kinetics of formation and physicochemical

Ako, K.; Nicolai, T.; Durand, D., Salt-induced gelation of globular protein aggregates: Structure and


23


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541

50.

542

Protein concentration and protein-exposed hydrophobicity as dominant parameters determining flocculation of

543

protein-stabilized oil-in-water emulsions. Langmuir 2013, 29, 11567-11574.

Delahaije, R. J. B. M.; Wierenga, P. A.; van Nieuwenhuijzen, N. H.; Giuseppin, M. L. F.; Gruppen, H.,

544


24

Page 25 of 32


545

Table 1. Molecular protein properties of β-lactoglobulin, ovalbumin and patatin as obtained from

546

the Swiss-Prot database (http://www.expasy.org).

547

Protein

Expasy code

Mw [kDa]

pI

#COOH/#NH2b

S-S bridges/free SH groups

σw [mC/m2]c

Rel. QH [-]d

β-lactoglobulin

P02754

18.4/36.8a

4.83

26/18

2/1

-21.9

1.00

ovalbumin

P01012

44.5

5.19

47/35

1/4

-24.2

0.19

patatin

P07745

40.0

5.25

43/32

0/1

-17.9

0.73

548

a

549

b

550

acid) and positively charged (arginine and lysine) groups in the primary sequence, respectively.

551

c

552

d

Molecular weight of a β-lactoglobulin dimer (native state under the selected conditions). #COOH and #NH2 are the maximum numbers of negatively charged (aspartic and glutamic

σw is the theoretical net surface charge density at pH 7. Rel. QH is the relative exposed hydrophobicity of the proteins as determined previously50.

553 554


25


Page 26 of 32

555

Table 2. Aggregation order of ovalbumin, β-lactoglobulin and patatin determined by different

556

methods. The variation reported for method 3.1-3.3 represent the standard deviation between R2

557

values obtained from fitting the individual conditions separately.

Determination of the aggregation order

Ovalbumin

β-Lactoglobulin

Patatin

1.0

1.5

1.0

2.1

2.5

2.9

(3.1) Linear fit of ln(Ct) in time (n = 1.0) (Ct ≥ 0.05C0)

R2 = 0.28 ± 0.32

R2 = 0.85 ± 0.13

R2 = 0.28 ± 0.30

(3.2) Linear fit of 1/√C in time (n = 1.5) (Ct ≥ 0.05C0)

R2 = 0.80 ± 0.13

R2 = 0.96 ± 0.04

R2 = 0.83 ± 0.09

(3.3) Linear fit of 1/C in time (n = 2.0) (Ct ≥ 0.05C0)

R2 = 0.95 ± 0.04

R2 = 0.93 ± 0.07

R2 = 0.97 ± 0.01

(1)

Concentration dependence of the th (Ct ≥ 0.5C0)

(2) Fit concentration in time using equations 1 and 2 (Ct ≥ 0.05C0)

558 559


26

Page 27 of 32


1

A - ova

0.9

0.8

0.8

Fraction C0 [-]

Fraction C0 [-]

1 0.9 0.7 0.6 0.5 0.4 0.3

0.7 0.6 0.5 0.4 0.3

0.2

0.2

0.1

0.1

0 0.0001

D - ova

0 0.01

1

100

10000

0.1

1

t/th [-] 0.9

10000

0.8

0.8

0.7 0.6 0.5 0.4 0.3

E - β-lg

0.7 0.6 0.5 0.4 0.3

0.2

0.2

0.1

0.1 0 0.01

1

100

10000

0.1

1

t/th [-]

10

100

1000

10000

Time [min]

1

1

C - pat

0.9

0.9

0.8

0.8

Fraction C0 [-]

Fraction C0 [-]

1000

1

B - β-lg

Fraction C0 [-]

Fraction C0 [-]

1

0.7 0.6 0.5 0.4 0.3

F - pat

0.7 0.6 0.5 0.4 0.3

0.2

0.2

0.1

0.1

0 0.0001

100

Time [min]

0.9

0 0.0001

10

0 0.01

1

100

10000

0.1

1

10

100

1000

10000

560 561

Figure 1. Fraction of non-aggregated protein (C0) as function of the time normalized by th (A-C)

562

and as function of the time (D-F) for ovalbumin (A and D), β-lactoglobulin (B and E) and patatin

563

(C and F). The markers represent the different conditions: Td - 10 °C (), Td (), Td + 5 °C

564

(), pH 5.0 (), pH 6.0 (), 5 g/L, 10 g/L, 40 mM NaCl and 90 mM NaCl () and the average

565

of all remaining conditions (Td - 5 °C, 2 g/L, 10 mM NaCl and all not indicated by the other

566

markers), with error bars indicating the standard deviation (). Solid lines in D-F are guides to

567

the eye. The solid lines in A-C represent the best fits (n = 2.1 for ovalbumin, n = 2.5 for β-

568

lactoglobulin, and n = 2.9 for patatin).

t/th [-]

Time [min]


27


Page 28 of 32

1000

th [min]

100

10

1 1

569

10

Concentration [g/L]

570

Figure 2. Concentration dependence of the time required to aggregate half of the proteins (th) for

571

ovalbumin (), β-lactoglobulin () and patatin () heated at Td - 5 °C. The solid line has a

572

slope of -0.5. The dashed line is a guide to the eye.

573


28

Page 29 of 32


574

Table 3. Half times (th) and aggregation rate (k) of the ovalbumin, β-lactoglobulin and patatin at

575

different heating conditions. k [L/g s] (n =2)a

th [min] Variable

C [g/L]

pH

I [mM]

T [°C]

Ova

β-Lg

Pat

Ovalbumin

β-Lactoglobulin

Patatin

Ref

2

7

10

Td - 5

8.7

223

10.8

9.64E-04

3.74E-05

7.71E-04

2

7

10

Td - 10

81

330

144

1.02E-04

2.52E-05

5.78E-05

2

7

10

Td

2.0

29.2

1.5

4.21E-03

2.85E-04

5.66E-03

2

7

10

Td + 5

1.1

16.0

0.5

7.87E-03

5.20E-04

1.60E-02

2

7

20

Td - 5

7.8

188

10.7

1.07E-03

4.42E-05

7.76E-04

2

7

50

Td - 5

8.7

93

9.9

9.63E-04

8.94E-05

8.38E-04

2

7

100

Td - 5

6.8

87

7.2

1.22E-03

9.63E-05

1.16E-03

1

7

10

Td - 5

9.9

255

11.1

1.69E-03

6.53E-05

1.50E-03

5

7

10

Td - 5

8.9

128

10.8

3.75E-04

2.59E-05

3.07E-04

10

7

10

Td - 5

8.0

84

13.3

2.08E-04

1.99E-05

1.26E-04

2

6

10

Td - 5

7.1

621

49.1

1.17E-03

1.34E-05

1.70E-04

2

5

10

Td - 5

4.8

1052

73

1.72E-03

7.92E-06

1.14E-04

T

I

C

pH

576 577

a

aggregation rate determined based on n = 2.

578


29

Page 30 of 32

A280 [mAU mL]


0.1

579

1

10

100

1000

10000

Time [min]

580

Figure 3. UV peak area of aggregated patatin (10 mM sodium phosphate buffer pH 7.0, 2 g/L,

581

Td - 5 °C) in time determined by size-exclusion chromatography. The markers represent samples

582

heated at different NaCl concentrations: 0 mM NaCl (), 10 mM NaCl () and 90 mM NaCl

583

().

584


30

Page 31 of 32


14

14

A - ova 5

12

5

12

10

10

8

8 6

I/I0 [-]

I/I0 [-]

D - ova

7 4 3,6 2 1,9-11

4 2

8

8 6

7 4 2 3,6

4 2

0 0

1200

2400

3600

4800

6000

7200

0

0.2

Time [s] 25

0.4

0.6

25

B - β-lg

E - β-lg

15

15

5

10 5

I/I0 [-]

20

I/I0 [-]

20

0 1200

2400

3600

4,8

4800

6000

2,7

5

1,3,6,9-11

0 0

7200

0.2

Time [s] 50

50

0.6

0.8

1

F - pat

45 40

35

35

30

30

5

I/I0 [-]

40

I/I0 [-]

0.4

Fraction aggregated protein [-]

C - pat 5

45

1

5

10

2 7 1,3,4,6,9-11 0

0.8

Fraction aggregated protein [-] 8

25

25

20

20

7

15

8

4,6 1-3,10,11 9

10 5 0

7

15

8

6 1-3 10 11

10

4

5

9

0 0

585

1,9-11

0

1200

2400

3600

4800

6000

Time [s]

7200

0

0.2

0.4

0.6

0.8

1

Fraction aggregated protein [-]

586

Figure 4. Normalized intensity (I/I0) of heated ovalbumin (A and D), β-lactoglobulin (B and E)

587

and patatin (C and F) in time (A-C) and as a function of the fraction aggregated protein (D-F).

588

The numbers represent the different conditions: (1) Td - 5 °C and 2 g/L, (2) 10 g/L, (3) 5 g/L, (4)

589

pH 6.0, (5) pH 5.0, (6) 10 mM NaCl, (7) 40 mM NaCl, (8) 90 mM NaCl, (9) Td - 10°C, (10) Td

590

and (11) Td + 5°C. The error bars indicate the standard deviation between the different

591

conditions with similar behavior.


31


592

Page 32 of 32

Table of Contents Graphic

593


32

Comparison of Heat-Induced Aggregation of ... - ACS Publications

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