-
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
-
......075..........
.. ,
:p....
100
cG)..o..Q.
50G)-.Q::so(/)c
oANS
95~" :;A- ----0 ---A 85" "~',
;,.'.,.,
"";,.'.'.'.'.'.,
"
:'.'.,.,.,., . I :
i:/:~..'
'.
~0
c4f2 4 6 8 10Hydrophobicity x 10-2
1.0
z
:.50)CG)....
'"
o 2ANS
.
0:" ," ." .
:h~: . . .
::I;J : :
: ',I.,
0'ii,...(D..... 95
\\ .. 0'A,
". 85'A- _" ~
75~'Q) ..01' ' 04 6 8
Hyd rophobicity x 10-210
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.
c
0 75....
"'b
4\... .
, '.. ., .
0'.'..' ... . .
: : ....~" ". "'n,
,
. .: c:i
/:,....95 o ,-ric .. .. "
o8~...~----A,"'"
75 ... 0 0- .....0O O M) O _ _10 20
IZ PI, mY
1.0
z
z:...
.5CJ)cG)....(/)
G)CJ
10 20IZ PI, mY
.00--- -. -_., -A- _. o..~:::..... ,
A\85
II
,
,
I
..
100
cG)..0..Q.
.! 50
.Q::s0(/)c
~
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
-
N ~6 \I 80 18 9 6.,..)(~"6(,)
"35.-~0J::.g-4... ~3"~J:
2UJZ 43 35 26 18 9c:( I 60 75
/ I. I /80 85 90 95
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
-
ReferencesBeveridge, T., .Jones, L. and Tung, M.A. 1984. Progel
and gel for-
matIon and reversibility of gelation of whey, soybean,and
albumen protein gels. 1. Agric. Food Chern. 32:307.
Dunkerley, 1.A. and Hayes, J.F. 1980. Characterization of
wheyprotein gels using a temperature gradient block. NewZealand 1.
Dairy Sci. 15:191.
Egelandsdal, B. 1980. Heat-induced gelling in solutions of
ovalbu-min. J. Food Sci. 45:570.
Ferry, J.D. 1948. Protein gels. Adv. Protein Chern.
4:2.Fothergill, L.A. and Fothergill, J.E. 1970. Thiol and disulfide
con-
tents of ovalbumin. C-terminal sequence and location ofdisulfide
bond. Biochem. J. 116:555.
Fujii, S. and Nakai, S. 1980. Optimization of data
transformationsfor linearization. Can. Inst. Food Sci. Techno!. J.
13: 188.
Gossett, P.W., Rizvi, S.S.H. and Baker, R.C. 1984.
Quantitativeanalysis of gelation in egg protein systems. Food
Tech-no!. 38(5):67.
Habeeb, A.F.S.A. 1972. Reaction of protein sulfbydryl groups
withEllman's reagent. Methods Enzymo!. 25:457.
Halwer, M. 1954. Disulfide cross-links in denatured ovalbumin.
J.Am. Chern. Soc. 76:183.
Hayakawa, S. and Nakai, S. 1985. Relationships of
hydrophobic-ity and net charge to the solubility of milk and soy
pro-teins. J. Food Sci. 50: 486.
Hegg, P-O. 1982. Conditions for the formation of heat-induced
gelsof some globular food proteins. 1. Food Sci. 47: 1241.
Hermansson, A-M. 1979. Aggregation and denaturation involvedin
gel formation. In: "Functionality and Protein Struc-ture". A.
Pour-El (Ed.). p. 81. ACS Symp. Series 92.Am. Chern. Soc.,
Washington, DC.
Hermansson, A-M. 1982. Gel characteristics-structure as related
totexture and waterbinding of blood plasma gels. J. FoodSci. 47:
1965.
Hickson, D.W., Dill, C.W., Morgan, R.G., Sweat, V.E., Suter,D.A.
and Carpenter, Z.L. 1982. Rheological propertiesof two heat-induced
protein gels. J. Food Sci. 47:783.
Can. Ins!. Food Sci. Technol. J. Vol. 18, No.4, 1985
Howell, N.K. and Lawrie, R.A. 1984. Functional aspects of
bloodplasma proteins. II. Gelling properties. J. Food
Techno!.19:289.
Kato, A, and Nakai, S. 1980. Hydrophobicity determined by
afluorescence probe method and its correlation with sur-face
properties of proteins. Biochim. Biophys. Acta624:13.
Kato, A., Nagase, Y., Matsudomi, N. and Kobayashi, K.
1983.Determination of molecular weight of soluble
ovalbuminaggregates during heat denaturation using low angle
laserlight scattering technique. Agric. BioI. Chern. 47:1829.
Li-Chan, E. 1983. Heat-induced changes in the proteins of
wheyprotein concentrate. J. Food Sci. 48:47.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.
1951.Protein measurement with the folin-phenol reagent. J.Bio!.
Chern. 193:265.
Nakamura, R., Sugiyama, H. and Sato, Y. 1978. Factors
contribut-ing to the heat-induced aggregation of ovalbumin.
Agric.Bio!. Chern. 42:819.
Ma, C. Y. and Holme, J. 1982. Effect of chemical modificationson
some physicochemical properties and heat coagula-tion of egg
albumen. J. Food Sci. 47:1454.
Shimada, K. and Matsushita, S. 1980. Thermal coagulation of
eggalbumin. J. Agric. Food Chern. 208:409.
Schmidt, R.H. and Morris, H.A. 1984. Gelation properties of
milkproteins, soy proteins, and blended protein systems.
FoodTechno!. 38(5):85.
Voutsinas, L.P., Nakai, S. and Harwalker, V.R. 1983.
Relation-ships between protein hydrophobicity and thermal
func-tional properties of food proteins. Can. Inst. Food
Sci.Techno!. J. 16:185.
Ziegler, G.R. and Acton, J .C. 1984. Mechanisms of gel
formationby proteins of muscle tissue. Food Techno!. 38(5):67.
Accepted May 13, 1985
Hayakawa and Nakai / 295
Contribution of Hydrophobicity, Net Charge and Sulfhydryl Groups
to Thermal Properties of Ovalbumin
