818 J. Agric. Food Chem., Vol. 27, No.

4, 1979 Lakshmi, Nandi

"^NH
1

1
—
hn*c-r
HOS^
LITERATURE CITED
\/ 1
Howe, R. K., Franz, J. E., J. Org. Chem. 39, 962 (1974).
h2sY-h2o o III T Kitamura, R., Suzuki, S., Yakugaku Zasshi 57, 659 (1937).
Kresze, G., Horn, A., Philippson, R., Trede, A., Chem. Ber. 98,
-
N 3401 (1965).
R-C —

Linser, H., “The Chemistry and Mode of Action of Plant Growth

ll H
RCN*S
Nss/"R ||

Substances”, Proceedings of a Symposium held at Wye College

(A) (B) (University of London), July 1955, Wain, R. L., Wightman, F.,
Ed., London: Butterworth, London, 1956, p 141.
Figure 3. Possible modes of formation of the 3,5-diphenyl- Mack, W., Angew. Chem., Int. Ed. Engl. 6, 1084 (1967).
1,2,4-thiadiazoles. Wegler, R., Chem. Pflanzenschutz-Schaedlingsbekaempfungs-
hitherto generally held opinion of the action of 2,6-di- mittel 5, 211 (1977).
chlorothiobenzamide (le) being due entirely to the for-
mation of 2,6-dichlorobenzonitrile (2e) is only of limited
validity. Received for review December 4,1978. Accepted March 6,1979.
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Studies on the Effect of Heat on the Dissociation, Denaturation, and Aggregation

of Sesame a-Globulin
T. S. Lakshmi and P. K. Nandi*1

The protein a-globulin, the major fraction of sesame seed (Sesamum indicum L.) proteins, coagulates
on heating. Dissociation, denaturation, and aggregation of the protein upon heating have been studied
by gel filtration, polyacrylamide gel electrophoresis, sedimentation velocity, pKapp of tyrosyl groups,
and fluorescence measurements. The addition of /3-mercaptoethanol does not reduce the extent of heat
coagulation. The reassociation of the heat denatured subunits through hydrophobic interaction results
in the formation of insoluble precipitate.

Sesame seed (Sesamum indicum L.) is a source of 6B-100 (Sigma) and urea (Sarabhai M. Chemicals) were
nutritionally important proteins due to their relatively high used. NaDodS04 (Hindustan Levers) was crystallized
methionine content. The protein upon heating results in twice from ethanol.
precipitation which restricts its use in certain food for- Heat coagulation experiments were carried out with
mulations, e.g., milk extender or beverage formulation. protein solution in Tris-HCl buffer of pH 8.6 at 98 °C for
Recently, we have reported the association-dissociation 20 min. The absorbance of the supernatant was measured
and denaturation behavior of the major constituent at 280 nm. The percentage of protein precipitated was
(65-70%) a-globulin of sesame protein in different so- determined by calculating the amount of protein present
lutions (Prakash and Nandi, 1976, 1977a,b,c, 1978; in the supernatant compared to the initial concentration
Lakshmi and Nandi, 1977,1978). In the present paper we of the protein solution. Protein concentration was cal-
report a study of the sequence and mechanism of heat culated using Elcm1% = 10.8.
aggregation of the protein. A Sepharose 6B-100 column 46 X 2.5 cm (bed volume,
MATERIALS AND METHODS Vt ~
200 mL), was used for gel filtration experiments. The
The protein a-globulin was isolated from sesame seeds gel was equilibrated thrice the bed volume of the column
with Tris-HCl buffer, 0.01 M, pH 8.6. The flow rate after
(Sesamum indicum L., white variety) following the pro-
cedure developed in this laboratory (Prakash and Nandi, loading the protein solution was adjusted to 18-20 mL/h
and the protein concentration in the fractions was de-
1978). The total protein extract in 1 M NaCl obtained termined by measuring the absorbance at 280 nm.
from defatted sesame flour was diluted 1:5.5 times with
distilled water when a-globulin with some other protein Polyacrylamide gel electrophoresis (PAGE) was carried
out in a Metrex gel electrophoresis unit using 0.02 M
fraction precipitated. The redissolution of the precipitate
in 1 M NaCl, followed by dilution as above, yielded a phosphate buffer at pH 7.5. A 10% gel in tubes having
7.5 X 0.5 cm dimensions was used. Protein samples (10
protein which was found to be homogeneous (~95%) by
Pg/pL) containing ~5% sucrose and 0.05% bromophenol
gel electrophoresis, sedimentation analysis, and DEAE- blue (indicator dye) were used, and electrophoresis was
cellulose chromatography (Prakash and Nandi, 1978).
carried out at a constant current of 3 mA/tube for 1 h and
Phosphate buffer prepared from reagent grade chemicals 40 min. The gels were stained for 45 min in 0.5% amido
and Tris (hydroxymethylaminomethane) obtained from
black in 7.5% (v/v) acetic acid, and destaining was carried
Sigma were used in most of the experiments. Sepharose out in 7.5% acetic acid solution.
Sedimentation velocity values were measured in a
Central Food Technological Research-Institute, My- Spinco Model E analytical ultracentrifuge equipped with
sore-570013, India. phase plate schlieren optics. A standard 12-mm dura-
xPresent address: Visiting Scientist, CEB, NIAMDD, luminum cell centerpiece was used. Plates were read on
Bethesda, Maryland 20014. a Gaertner microcomparator and s20,w values calculated

0021-8561/79/1427-0818901.00/0 © 1979 American Chemical Society

Study of Sesame «-Globulin J. Agric. Food Chem., Vol. 27, No. 4, 1979 819

Cone, of Protein mg/ml

Figure 1. Effect of temperature on the percentage of protein
precipitation using varying concentrations of protein in the
solution. Temperature, 98 °C; time of heating, 20 min.
Figure 2. Gel filtration pattern of (1) control a-globulin and (2)
heated protein in Sepharose 6B-100 gel column, dimension 46 X
(Schachman, 1959). The percentage of the different 2.5 cm; bed volume (Vj, 200 mL in 0.1 M Tris-HCl buffer of pH
components was estimated from enlarged tracings of 8.6.
sedimentation velocity patterns.
Spectrophotometric titration of the phenolic groups in
the protein was carried out by measuring the absorbance
at 295 nm as a function of pH (Donovan, 1973; Mihalyi,
1968). A protein solution of 0.05% (0.45 OD at 280 nm)
and the supernatant of the heated protein solution in 0.1
M Tris of pH 8.6 were used for the titration. The pH was
adjusted by adding 10% NaOH to the protein which was
initially present in Tris buffer of pH 8.6. Possible turbidity
corrections were made by subtracting absorbance at 330
nm from 295 nm absorbance (Mihalyi, 1968).
Fluorescence was measured in a Perkin-Elmer Hitachi
fluorescence spectrophotometer using 0,005% protein
solution in 0.1 M Tris-HCl of pH 8.6. The excitation
wavelength was 280 nm.
The number of SH groups in a-globulin was estimated Figure 3. PAGE pattern of (1) control a-globulin, (2) a-globulin
using Ellman’s reagent [5,5,-dithiobis(2-nitrobenzoic acid), heated for 10 min, and (3) a-globulin heated for 30 min in 0.01
Beveridge et al„ (1974)]. An aliquot (0.2 mL) of 1% M phosphate buffer of pH 7.5.
protein solution was added to 2.5 mL of buffer or 8 M urea
and the color was developed by addition of 0.02 mL of The protein a-globulin has a sedimentation coefficient
Ellman’s reagent. The absorbance was measured at 412 of 13S in Tris-HCl buffer, 0.1 M, pH 7.0 (Lakshmi and
nm in a colorimeter and the moles of SH/mole of protein Nandi, 1977). At pH 8.6, Tris-HCl, the protein dissociates
was calculated from (~20%) to an 8S component (Figure 4a,b) at 28 °C. The
protein solution at 40 °C indicated a decrease in the
[1/(1-36 X 104)]A4I2(D/C) concentration (10%) of the 8S component with a con-
where 1.36 X 104 is the molar absorptivity of the complex, comitant increase (90%) of 13S component. Upon cooling
A412 is the absorbance at 412 nm, D is the dilution factor,
the solution to 28 °C, the same sedimentation pattern as
and C is the sample concentration in milligrams/milliliter. observed with the unheated protein solution was observed.
The number of SH groups in the absence and presence of This indicated that association-dissociation of a-globulin
urea were 1 and 7 mol of SH groups/mole of protein, up to 40 °C was reversible. The protein heated to 50 °C
respectively. showed two components sedimenting with 15S and 8S
(Figure 4c) value. The pattern did not change up to 60
RESULTS AND DISCUSSION °C (Figure 4d). The protein solution heated at 80 °C for
The protein solution when heated at 98 °C for 20 min 5 min showed a higher percentage (95%) of the 15S
results in 92% precipitation (Figure 1) and the amount of component. In addition, the presence of a dissociated
precipitation was independent of the initial concentration fraction having 3S value was observed (Figure 4e). The
of the protein within the range studied (4.6-27.8 mg/mL). same solution heated at 80 °C for 10 min showed four
The protein in 0.1 M Tris-HCl buffer of pH 8.6 in the components with sedimentation values of 17, 15, 10 and
Sepharose 6B-100 column elutes near the void volume of 3S (Figure 4f). The solution heated at 80 °C for 15 min
the gel (~68 mL), Supernatant of the heated protein showed a fast moving component sedimenting with 77S
solution shows (Figure 2) four components eluting at ~25, value (Figure 4g) together with a lower aggregate of 26S
~50, ~90 , and ~150 mL in addition to the peak ap- value and the dissociated products of 6 and 3S (Figure 4h).
pearing near 68 mL. The results indicate that in the Heating for 30 min at 80 °C resulted in the disappearance
heated protein solution both aggregated and dissociated of the fast moving components. The solution showed a
products are present. component of 15S, constituting ~80% of the total, and
The supernatant protein solution (98 °C, 10 min) two other components, 8 and 3S (10% each) (Figure 4i).
subjected to PAGE showed (Figure 3) six major and one The supernatant of the protein solution heated to 98 °C
minor bands; the presence of a high polymer and several for 20 min showed the same pattern as that of the protein
dissociated components could be observed. The solution heated at 80 °C for 30 min (Figure 4j).
on prolonged heating (98 °C, 30 min) indicated a decrease The apparent pKint value of tyrosyl groups of sesame
in the amount of the polymeric form. The mobility of the a-globulin in 0.1 M 'I ris is 11.0 which indicates that tyrosyl
dissociated products was nearly the same in the two groups are abnormal and are not in contact with the
samples. aqueous environment (Donovan, 1973). The supernatant
820 J. Agric. Food Chem., Voi. 27, No. 4, 1979 Lakshmi, Nandi

Figure 4. Effect of temperature on the sedimentation velocity pattern of a-globulin in 0,1 M Tris-HCl buffer, pH 8.6. (a) a-Globulin
in 0.05 M Tris-HCl buffer of pH 7.2, a-globulin at pH 8.6 (b) at 28 °C, (c) at 50 °C for 20 min, (d) at 60 °C for 20 min, (e) at 80 °C
for 5 min, (f) at 80 °C for 10 min, (g) at 80 °C for 15 min (27150 rpm), (h) at 80 °C for 15 min, (i) at 80 DC for 30 min, (j) at 98 °C
for 20 min. Sedimentation proceeds from left to right. A speed of 59780 rpm was used unless otherwise indicated. Temperature,
27 °C.

of the heated protein solution (98 °C, 30 min) showed a

pKint value of 10.6 (Figure 5). This suggested that the
tyrosyl groups are experiencing a more polar environment
as a consequence of denaturation upon heating.
The emission maximum of a-globulin when excited by
280-nm light is at 325-328 nm. This arises from the
tryptophan groups embedded in the nonpolar environment
of the protein and, the tyrosyl groups do not contribute
to the observed fluorescence (Lakshmi and Nandi, 1977).
The fluorescence spectrum of the protein solution heated
at temperatures up to 50 °C showed neither a change in
the emission maximum nor intensity. The solution at 57 Figure 5. Spectrophotometric titration to determine the apparent
°C, however, showed considerable decrease in intensity pKtot value of the tyrosyl groups of a-globulin at room temperature
with a slight red shift to 333 nm. Heating at 80 °C results (28 °C) and supernatant of the heated protein solution at 98 °C.
(1) pKtat value of heated a-globulin, 10.6; (2) pK^t value of control
in further decrease in the intensity and a shift in the
a-globulin, 11.0.
maximum to 340 nm. The samples heated at 88 and 97
°C showed further decrease in the intensity and shift in environment. This has resulted from the unfolding, i.e.,
the emission maximum to 345 nm (Figure 6). This shift denaturation of the protein molecule at high temperature.
of the emission maximum toward red arises from the The results obtained from the above experiments suggest
presence of the tryptophan groups in a polar aqueous that a-globulin upon heating undergoes dissociation,
Study of Sesame a-Globulin J. Agric. Food Chem., Vol. 27, No. 4, 1979 821

Figure 8. Effect of increasing concentration of NaDodS04 the

Figure 6. Fluorescence spectra of a-globulin at room temperature
(28 °C) and protein solution heated at different temperatures. heat coagulation pattern of a-globulin.
Time of heating, 20 min; excitation, 280 nm.
protein. In the denatured state, the favorable interaction
of the charged and polar groups with water (resulting from
charge solvation and hydrogen bonding ability with water,
respectively) would not be able to overcome the unfa-
vorable interaction of the nonpolar groups with water
which leads to the aggregation of the heat denatured
protein. Further, the electrostatic repulsion between the
different protein subunits as they carry overall negative
charges at the experimental pH 8.6 is not sufficient to
prevent aggregation of the denatured sesame a-globulin
upon heating.
The present results show that sesame a-globulin un-
dergoes dissociation, denaturation, and aggregation upon
heating. The aggregate does not dissolve upon cooling.
This apparently “irreversible” aggregation is always po-
tentially reversible since no covalent bond formation has
taken place in the process. But the activation enthalpy
barrier for dissociation of the aggregate is high, so that with
the decrease in temperature, refolding into original protein
Figure 7. Effect of increasing concentrations of urea on the will be a very slow process. This is the reason why, even
percent precipitation of protein upon heating at 98 °C for 20 min. upon cooling, the aggregate cannot dissolve but can be
dissociated by urea, amides (Lumry and Biltonen, 1969),
denaturation, and aggregation. The formation of higher and sodium dodecyl sulfate solution.
aggregates is endothermic in nature. This would suggest
that, upon heat denaturation, the newly exposed nonpolar LITERATURE CITED
groups of the protein due to their unfavorable interaction Beveridge, T., Toma, S. J., Nakai, S., J. Food Sci. 39, 49 (1974).
with the solvent water tend to come together, leading to Catsimpoolas, N., Funk, S. K., Meyer, E. W., Cereal Chem. 47,
aggregation. In addition, the polymerization process may 331 (1970).
also result from the formation of S-S linkages between the Donovan, J. W., Methods Enzymol. 27, 525 (1973).
polypeptide chains as a consequence of the oxidation of Lakshmi, T. S., Nandi, P. K., Int. J. Pept. Protein Res. 10, 120
the newly exposed SH groups (see Materials and Methods (1977) .

section) of the heat denatured protein (Tanrord, 1968, Lakshmi, T. S., Nandi, P. K., Int. J. Pept. Protein Res. 12, 197
(1978)
1970). However, the addition of /3-mercaptoethanol at
.

room temperature precipitated a-globulin and there was

Lumry, R., Biltonen, R., in “Structure and Stability of Biological
Macromolecules”, Timasheff, S. N., Fasman, C. D., Ed., Marcel
no prevention of heat coagulation by the reagent. Cat-
Dekker, New York, 1969, p 65.
simpoolas et al. (1970) observed that this reagent also did Mihalyi, E., Biochemistry 7, 208 (1968).
not prevent the heat coagulation of the soybean glycinin Prakash, V., Nandi, P. K., Int. J. Pept. Protein Res. 8, 385 (1976).
and concluded that polymerization of the heat denatured Prakash, V., Nandi, P. K., Int. J. Pept. Protein Res. 9, 97 (1977a).
protein does not take place through S-S linkage. The Prakash, V., Nandi, P. K., J. Biol. Chem. 252, 240 (1977b).
prevention of heat coagulation of a-globulin in urea, viz. Prakash, V., Nandi, P. K., Int. J. Pept. Protein Res. 9, 319 (1977c).
100% in 8 M urea (Figure 7) and anionic detergent sodium Prakash, V., Nandi, P. K., J. Agric. Food Chem. 26, 320 (1978).
Schachman, H. K., in “Ultracentrifugation in Biochemistry”,
dodecyl sulfate above 2 X 10“3 M (Figure 8), would suggest Academic Press, New York, 1959, p 75.
that the aggregation process of heat denatured sesame
Tanford, C., Adv. Protein Chem. 23, 122 (1968).
a-globulin takes place predominantly by the hydrophobic Tanford, C., Adv. Protein Chem. 24, 2 (1970).
interaction of the newly exposed nonpolar groups.
The amount of charge and number of polar groups are
the same in both the native and denatured state of the Received for review June 19, 1978. Accepted February 21,1979.