Can. Insf. Food Sci. Technol. J. Vol. 18, No.4, pp. 290-295, 1985

RESEARCH

Contribution of Hydrophobicity, Net Charge andSulfhydryl Groups to Thermal Properties of Ovalbumin

Shigeru Hayakawa and Shuryo NakaiDept. of Food Science

Univ. of British ColumbiaVancouver, B.C.

V6T 2A2

AbstractCoagulability and gel strength of ovalbumin measured after

heating 0.5070 and 5% solutions, respectively, were correlated toanilinonaphthalenesulfonate hydrophobicity (ANS), zeta potential(ZP) and sulfhydryl content (SH). Significant correlation to coagul-ability was obtained with the regression equation: [coagulabil-ity] = 0.476 ANS - 0.000404 ANS2 - 0.0137 ANS.ZP - 4.77 (R2= 0.794, P

of Lowry et al. (1951). Coagulability was calculatedfrom the following equation:

Ten mL of 5OJo ovalbumin solution in a sealed vial(2.3 cm id) was heated under specified conditions andcooled immediately to room temperature. Gel strengthof the heated sample was determined with an InstronUniversal Testing Instrument model 1122 (InstronLimited, High Wycombe, England) using a 0 - I kgload cell with an electronic 10: I scale expansion. Theinstrument was operated at a crosshead speed of 50mm/min. Gel strength was expressed as the force inNewtons applied to a cylindrical probe (7.9 x 150 mm)when the surface yield point was reached. Recordingof the load was continued until a depth of 15 mm wasreached.

Coagulability = 100(1 - Ps/Pt)where: Pt is protein in solution before heating.

Gel strength

(I)

8.0. Measurement was carried out at an applied poten-tial difference of 150 V. The electrophoretic mobilitywas automatically converted to zeta potential, and theabsolute value of zeta potential (ZP) in mV was usedin regression analysis.

Total sulfhydryl groupsTotal sulfhydryl (SH) groups were determined using

Ellman's reagent (5,5' -dithiobis-2-nitrobenzoic acid),according to Habeeb (1972). Coagulum in the heated0.5% or 5% ovalbumin solution was solubilized in0.086 M Tris-glycine buffer (pH 8.0) containing 2%sodium dodecyl sulfate and 0.004 M EDTA. To a 3mL aliquot of the sample solution was added 0.03 mLof Ellman's reagent solution (4 mg/mL). The absor-bance was read at 412 nm. Sample and reagent blankswere included for subtraction. A molar extinctioncoefficient of 1.36 x 104 M-Icm-I was used for cal-culating Il moles of SH/g of protein.

Hydrophobicity

Hydrophobicity was determined using hydrophobicfIuoresence probes, l-anilino-8-naphthalene sulfonate(ANS) and cis-parinaric acid (CPA). Measurementswere performed according to the method of Kato andNakai (1980) with slight modifications. Ovalbuminsolutions heated at the protein concentration of 0.5%were diluted serially with phosphate buffers describedabove (pH 5.5 - 8.0) to obtain protein concentrationsranging from 0.004 to 0.02 %. Then, 10 ilL of ANS(8.0 mM in 0.01 M phosphate buffer, pH 7) or CPA(3.6 mM in absolute ethanol containing equimolarbutylated hydroxytoluene) was added to 2 mL of sam-ple solution. Fluoresence intensity (FI) was measuredwith a Aminco-Bowman spectrophotofluorometer,No. 4-8202 at excitation wavelengths of 390 nm and325 nm, and emission at 470 nm and 420 nm for ANSand CPA, respectively. The FI reading was stan-dardized by adjusting the reading of the fluorometerto 30% full scale for ANS in methanol and 70% fullscale for CPA in decane. The net FI at each proteinconcentration was determined by subtracting FI ofeach protein solution without probe from that withprobe. The initial slope of the FI versus protein con-centration (%) plot, which was calculated by linearregression analysis using a Monroe 1880 programma-ble calculator, was used as an index of the proteinhydrophobicity.

Net chargeNet charge was measured with a particle microelec-

trophoresis apparatus (Pen Kern, Laser Zee Model501, Bedford Hills, NY). Protein particle suspensionwas prepared by homogenizing the mixture of 0.1 %protein solution diluted from 0.5% heated ovalbuminsolutions and 3,3' -dimethyl biphenyl (100:3) using aBrinkmann Polytron at 3000 rpm for 30 sec and thendiluted 40 fold in phosphate buffer with a specific con-ductance of 0.8 m mhos and pH ranging from 5.5 to

Can. lns(. Food Sci. Techno!. J. Vol. 18, No.4, 1985

Statistical analysis

Curve fitting was performed with a Monroe 1880programmable calculator according to Fujii and Nakai(1980). Backwards stepwise multiple regression anal-ysis was carried out with the UBC Triangular Regres-sion Package, while contour surface plots were gener-ated by using the UBC Surface Visualization Routinesprogram, with an Amdahl 470 V/8 computer.

Results and DiscussionIt is well known that ovalbumin is a heat-sensitive

protein and aggregates upon prolonged heating. Ferry(1948) suggested the following two step process forheat-induced aggregation: native protein - denaturedprotein - aggregated protein. Ma and Holme (1982)also proposed the thermocoagulation process of eggalbumen as follows: native monomer - denaturedmonomer - soluble aggregate - gel or coagulum.Aggregation is a general term representing protein-protein interaction (Hermansson, 1979). Coagulationis a random aggregation of denatured proteinmolecules (Hermansson, 1979), and the coagulum maysettle out of solution. In the present work, the extentof coagulation (coagulability) was shown as the per-centage of insoluble protein after centrifugation. Gela-tion involves the formation of the three-dimensionalnetwork (Hermansson, 1979) which can be assessed asgel strength. Coagulability and gel strength were deter-mined at protein concentrations of 0.5% and 5%,respectively. Ovalbumin heated at 0.5% formed no geland most of the ovalbumin samples heated at 5%formed gels which did not sediment by centrifugation.Therefore, coagulability and gel strength heated at0.5% and 5%, respectively, were used for regressionanalysis.

Figures 1 and 2 show changes in ANS hydropho-bicity in relation to coagulability and gel strength,respectively. Coagulability increased sharply in solu-tion with ANS hydrophobicity exceeding 350, whilegel strength was greatest at ANS hydrophobicity of

Hayakawa and Nakai / 291

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Fig. I. Coagulability of heated ovalbumin as a function of ANShydrophobicity. (e) unheated, (O)heated at 75C, (l':.) 85C,and (0) 95C for 15 min at protein concentration of 0.5010.From left to right datapoints the pH decreased: 8.0, 7.5,7.0,6.75,6.5,6.0 and 5.5

Fig. 2. Gel strength of heated ovalbumin as a function of ANShydrophobicity. (e) unheated, (0) heated at 75C, (l':.)85C, and (0) 95C for 15 min at protein concentrationof 5%. From left to right datapoints the pH decreased: 8.0,7.5,7.0,6.75,6.5,6.0 and 5.5.

around 300 for samples heated at all temperatures.These results suggest that thermal coagulation of oval-bumin is in part due to hydrophobic interaction.However, excessive hydrophobicity of proteindecreases gel strength. When hydrophobicity of heatedovalbumin was measured with cis-parinarate, theresults were almost the same as ANS hydrophobicity.The aromatic hydrophobicity determined by using

ANS may playa more important role in protein solu-bility than the aliphatic hydrophobicity determined byusing cis-parinarate (Hayakawa and Nakai, 1985).Therefore, it would seem reasonable to use ANShydrophobicity as a parameter representing hydropho-bicity in the present work, since coagulation and gela-tion are both closely related to solubility.

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Fig. 3. Coagulability of heated ovalbumin as a function of zetapotential (ZP). (e) unheated, (0) heated at 75C, (l':.) 85C,and (0) 95C for 15 min at protein concentration of 0.5%.From left to right datapoints the pH increased: 5.5, 6.0,6.5, 6.75, 7.0,7.5 and 8.0.

Fig. 4. Gel strength of heated ovalbumin as a function of zeta poten-tial (ZP). (e) unheated, (0) heated at 75C, (l':.) 85C, and(0) 95C for 15 min at protein concentration of 5%. Fromleft to right datapoints the pH increased: 5.5, 6.0, 6.5, 6.75,7.0, 7.5 and 8.0.

292 / Hayakawa and Nakai Can. Ins!. Food Sci. Technol. J. Vol. 18, No.4, 1985

Fig. 5. Gel strength of heated ovalbumin as a functin of total sulf-hydryl (SH) content. (e) unheated, (0) heated at 75C, (6)85C, and (0) 95C for 15 min at protein concentrationof 5%. Regression equation: yO.2 = 19.04 - 0.201 x(r = 0.875, P

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Tota I SH, J,lM/gFig. 7. Response surface contour of gel strength as functions of

ANS hydrophobicity and total sulfhydryl (SH) content. Gelstrength was shown as the force in Newton x 1()2.

gelation than for coagulation. Egelandsdal (1980) sug-gested that ovalbumin gelation was governed primar-ily by electrostatic forces. When zeta potential wasused as a sole function of gel strength measured at pHranging from 5.5 to 8.0, gel strength was affected bynet charge (Figure 4).

It is possible that decreases in sulfhydryl content arealso responsible for gelation. Involvement of sulf-hydryl groups in gelation of proteins (Voutsinas et al.,1983) and egg white proteins (Shimada and Mat-sushita, 1980; Ma and Holme, 1982) has beenreported. Ovalbumin is one of the relatively few pro-teins containing both thiol groups (4 moles) anddisulfide groups (1 mole) in the molecule (Fothergilland Fothergill, 1970). Ovalbumin can be polymerizedby intermolecular sulfhydryl-disulfide exchange dur-ing heating (Halwer, 1954). Less than I mole SH/moleof ovalbumin was converted to disulfide bonds (Figure5). Intermolecular sulfhydryl-disulfide exchange ena-bles ovalbumin to form simple linear aggregatesbecause the protein contains one mole of readily avail-able disulfide bond. Even though oxidized sulfhydrylgroups are less than one mole, a three dimensional net-work structure can be formed by extra disulfide bondsformed upon heating. Therefore, the conversion ofsulfhydryl groups to disulfide bonds as well as inter-change of sulfhydryl-disulfide groups may be impor-tant for gelation. However, Hegg (1982) indicated thatthere was no correlation between disulfide or sulf-hydryl content and gel-forming ability of globular pro-teins. On the contrary, Voutsinas et al. (1983) foundthat gelation was significantly (P

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Accepted May 13, 1985

Hayakawa and Nakai / 295

Contribution of Hydrophobicity, Net Charge and Sulfhydryl Groups to Thermal Properties of Ovalbumin