Biochimica et Biophysica Acta, 701 (1982) 237-241 237

Elsevier Biomedical Press

BBA 31059

THERMAL DENATURATION OF GENETIC VARIANTS OF THE KUNITZ SOYBEAN TRYPSIN

INHIBITOR

JAMES E. SANDERSON, ROBERT C. FREED and DALE S. RYAN *

Department of Food Science, 1234 Babcock Hall, University of Wisconsin, Madison, Madison, 14/153706 (U.S.A.)

(Received June 26th, 1981)

(Revised manuscript received September 25th, 1981)

Ke.r words: To'psin inhibitor; Heat stability; Genetic variant; (Kunitz soybean)

The thermal denaturation of Kunitz soybean .trypsin inhibitor is not a simple two-state process. A
low-temperature readily reversible transition with an enthalpy of 10-15 kcal/mol is followed by a higher-
temperature non-readily reversible transition with a much larger enthaipy (approx. 100 kcal/mol). The
genetic variants of soybean trypsin inhibitor have different thermal stabilities. The transition temperature for
the high-temperature thermal denaturation of SBTI-Ti ~ is 64°C. The transition temperature for SBTI-Ti b is
62°C and for SBTI-Ti c it is 57°C.

Introduction vide some interesting insights into the mechanism

Three genetic variants of soybean trypsin in-
hibitor have been identified [1-5]. These variant
forms give bands of different mobility on discon-
tinuous polyacrylamide gel electrophoresis and
have been designated Ti a, Ti b and Ti c [5]. A fourth
soybean variant, designated ti, apparently contains
none of the Kunitz soybean trypsin inhibitor [5-6].
The most common variant is Ti a which is present
in 88.8% of 3039 tested soybean varieties in the
USDA soybean germplasm collection. The Ti b, Ti c
and ti alleles were found in 10.9, 0.3 and 0.06%of
the tested varieties, respectively [7]. The associa-
tion equilibrium constant of the soybean trypsin
inhibitor variants for trypsin are 2. 109M-I, 2.
107M -I and 3-101°M -l for Ti a, Ti b and Ti c,
respectively [8]. These large differences in affinity
for trypsin suggest that these molecules might pro-
of trypsin inhibition.
The Kunitz soybean trypsin inhibitor is unusu-
ally resistant to thermal a n d chemical denatura-
tion. In 6 M guanidinium chloride soybean trypsin
inhibitor undergoes a remarkably slow unfolding
which requires 2 weeks to reach completion [9]. In
the presence of 9 M urea only slight changes in the
structure of the molecule are observed [10]. Ther-
mal denaturation of the molecule at neutral pH
does not take place until temperatures exceed 60°C
[11]. There are no unusual structural features of
the molecule which can readily explain this re-
markable stability [12]. The present study of the
thermal stability of genetic variants of soybean
trypsin inhibitor was begun in the hope of eventu-
ally identifying those structural features of the
molecule which are of particular importance for
thermal stability.

Materials and Methods

* To whom correspondence should be addressed at: 4051
Bethel Drive, No. 39, St. Paul, MN 55112, U.S.A. Glycine max. v. Steele soybeans (lot LA 1-4)
Abbreviations: SBTI, soybean trypsin inhibitor; TAME, tosyl- were purchased from Olds Seed Co., Madison, Wl.
arginyl methyl ester. Varieties T-245 and PI 246.367 were obtained

0167-4838/82/0000-0000/$02.75 ~ 1982 Elsevier Biomedical Press

238
from Dr. T. Hymowitz, Department of Agronomy,
University of Illinois, Urbana, IL. Trypsin from
bovine pancreas (Type XI), soybean trypsin in-
hibitor (type I-S), and p-tosyl-L-arginine methyl
ester-HCl (TAME) were purchased from Sigma
Chemical Co., St. Louis, MO.
Isolation of Kunitz soybean trypsin inhibitor.
Kunitz soybean trypsin inhibitor was isolated from
each soybean variety and from a commercial pre-
paration of Ti a using ion-exchange column chro-
matography as previously described [8]. Discon-
tinuous polyacrylamide gel electrophoresis of each
purified inhibitor showed a single band [8].
Thermal denaturation of soybean trypsin inhibi-
tor. Temperature-difference spectral measurements
were carried out on soybean trypsin inhibitor using
the following apparatus: a Beckman Model 25
Spectrophotometer with a thermostat-controlled
cell holder was used; water at 20°C from a re-
frigerated Haake water bath was circulated through
the cell holder to maintain a constant temperature
in the reference cuvette. A savant water-jacketed
cuvette was used for the samples. Temperature in
the sample cuvette was varied by pumping water
from a second water bath. Temperatures of solu-
tions in both cuvettes were continuously moni-
tored using copper-constantan thermocouples.
Soybean trypsin inhibitor solutions were made
by dissolving protein in 0.3 M KC1/0.02 M potas-
sium phosphate buffer adjusted to pH 6.5. Protein
concentrations were determined by measuring ab-
sorbance at 280 nm and using a molar extinction
coefficient of 9.44 [13]. Final concentrations of
soybean trypsin inhibitor were near 1.6 mg/ml.
Solutions were filtered through a 0.45-micron Mil-
lipore filter to remove particulate matter.
Samples were heated at approx. 1°C/min until
a maximum temperature of approx. 85°C was
observed. Changes in absorbance were recorded
every 5°C early in the experiment and at 1- or
2-degree intervals in the range of rapid absorbance
changes.
Esterase activity assay for trypsin inhibitor. The
esterase activity of trypsin was measured with an
assay similar to the one described by Walsh [141
using TAME. Details of this assay appeared in a
previous paper [8].
Results and Discussion
The thermal stability of three genetic variants
of the Kunitz soybean trypsin inhibitor was ex-
amined by monitoring the inhibitory activity of
the molecules against trypsin after exposure to
elevated temperatures for varying periods of time.
As can be seen in Fig. I, the stabilities of the
molecules are noticeably different at 60°C in
0.08 M Tris/0.02M CaCI 2, pHT.0. Ti a is quite
stable under these conditions but both Ti b and Ti c
lose most of their inhibitory activity at a fairly
rapid rate. These differences are less pronounced
but are still evident at 67°C (Fig. 2) where de-
naturation of all three molecules is quite rapid. It
should be noted that not all of the inhibitory
activity of the molecules was lost during these
experiments. 20 to 30 percent of the activity of the
inhibitor remained even after prolonged incuba-
tion at the elevated temperature. It could be that
the thermally denatured molecule retains some
inhibitory activity against trypsin but more proba-
bly these results suggest that a significant per-
centage of the inhibitor molecules exist after ther-
mal denaturation in a form which readily refolds
to the native conformation when the temperature
is reduced. Some additional evidence which sup-
ports this suggestion was obtained by a more
lOG
8c
Z
tu
er
>-
mZ
"1-
4G r
20 I I I
0 10 20 30
TIME (MINUTES)
Fig. I. Heat inactivation of soybean trypsin inhibitor variants
in 0.08 M Tris/0.02 M CaCI 2, pH 7.0. at 60°C. The loss in
inhibitor activity was calculated from changes in the extent of
inhibition of trypsin under conditions where inhibition should
be stoichiometric. See text for details. 0 . Ti~: A . TiB: I . TiL
 239

IOO' I00 ' I ' I ' 1 ' I '-~ F '~f

v 8O 80 ! -
Z

~60
>-
• ~ 60-

p- /+Or- ~, Ttr=63°C -
4o

I
_z 2~
=7
0
I
2
I
4 6
L
8
L
I0
] I
12
I
14
I
16
1
18
I
20
20 30 ~3 50 fW3 70
TIME ( M I N U T E S )
TEMPERATURE (%)
Fig. 2. Heat inactivation of soybean trypsin inhibitor variants Fig. 3. Thermal denaturation of Ti b in 0.3 M KCI/0.02 M
in 0.08 M Tris/0.02 M CaCI 2. pH 7.0, at 67°C. The loss in potassium phosphate, pH 6.5.
inhibitor activity was calculated from changes in the extent of
inhibition of trypsin under conditions where inhibition should
be stoichiometric. See text for details. O, Tia; A Tib; I , Ti c.

of this kind is commonly defined as the tempera-

careful examination of the thermal unfolding of
the molecule.
Thermal denaturation of soybean trypsin in-
hibitor is accompanied by large changes in the
absorbance spectra of the molecule. Three 'peaks'
which increase with increasing temperature are
present with maxima at 245nm, 288nm and
298 nm. There is, in addition, a 'valley' between
260 and 280 nm which decreases with increasing
temperature. The wavelength chosen for further
difference spectra measurements (298 nm) is the
same as that used by Wu and Scheraga [11] and
corresponds to the high-wavelength maximum in
the thermal difference spectra. Our data, therefore,
probably reflect environmental changes experi-
enced by the aromatic amino acids in the mole-
cule.
The effect of increasing temperature on the
absorbance of Ti b at 298 nm is shown in Fig. 3 and
is typical of the curves obtained for the three
varieties. The rate of heating in our experimental
system was fairly constant over the period of the
experiment. The absorbance of the solution at
298 nm, however, increased gradually at the be-
ginning of the experiment, then.increased rapidly
at the temperature range of approximately 55 to
65°C and then stabilized again at higher tempera-
tures. The transition temperature for a transition
ture at which the percent of maximum absorbance
change equals 50. The shapes of these curves sug-
gest that at least two distinguishable processes are
taking place. Even at the lowest temperature,
changes in the absorbance of the molecule can be
observed. This lower-temperature transition is,
however, not readily separable from the more pro-
nounced transition which begins at higher temper-
atures.
Transition temperatures were obtained from
multiple runs with each molecule and these data
are presented in Table I. The transition tempera-
ture obtained from four runs on Ti a was 64.4-+
0.5°C. This is in fairly close agreement with a
measurement of 63.8°C reported by Wu and
Scheraga [11] using the same buffer, ionic strength
TABLE I
TRANSITION TEMPERATURES FOR THERMAL DE-
N A T U R A T I O N OF SOYBEAN TRYPSIN INHIBITOR
(SBTI)
Sample Temperature
(°C)
SBTI-Ti a 64.4-+0.5
SBTI-Ti b 62.3 -+ 1.2
SBTI-Ti c 57.4-'- 2.3
240

i I i I I I i I l
5.0 is equal to the percent of total absorbance change
at that temperature times the protein concentra-
4.0
tion. The concentration of the native species at
3.0
this temperature is 100 minus the concentration of
the denatured species. Fig. 4 shows a typical Van't
2.0 Hoff plot from our data. Thermodynamic data
from Van't Hoff plots for Ti a, Ti b and Ti c are
z 1.o summarized in Table II. Although resolution of
z the Van't Hoff curves into two distinct lines is
J 0
somewhat arbitrary, it can be seen that there are at
least two separate events occurring during thermal
-1.C
denaturation of soybean trypsin inhibitor. The first
-2.(
(low-temperature) event has an enthalpy in the
range of 11-14 kcal/mol and an entropy of 30-40
-3.( cal/mol per K. The second event, which occurs in
the transition temperature range, has a much higher
-4.0 ' I = I I , I I I I enthalpy with values more typical for protein de-
2.8 2.9 3.0 3.1 3.2 3.3 34
I031T naturation (85-127 kcal/mol). The enthalpy value
for Ti a (97-+ 24 kcal/mol) is consistent with the
Fig. 4. V a n ' t H o f f plot of thermal d e n a t u r a t i o n of Ti b.
value of 1 1 0 - 5 kcal/mol reported by Donovan
and Beardslee [15] from calorimetric studies. The
presence of two separate and distinct regions of
and pH. The average values for transition temper- the Van't Hoff plot for soybean trypsin inhibitor
atures for Ti b and Ti c were slightly lower than that suggests that thermal denaturation of soybean
for Ti a. The value for Ti b is roughly 2°C less than trypsin inhibitor is not a simple two-state process.
that for Ti a and the value for Ti c is 7°C less than Significant levels of at least one intermediate form
that for Ti a. These observations are consistent of the molecule must accumulate during thermal
with the relative stabilities observed using the en- denaturation.
zymatic assay. The reversibility of the thermally induced
Van't Hoff plots of the thermally induced ab- changes in the absorbance spectra of soybean
sorbance changes in the three variants of soybean trypsin inhibitor was examined to see if the low-
trypsin inhibitor were made by plotting In K against temperature transition differed from the high-
1/T where K is the molar ratio of 'denatured' to tempeature transition. A solution of Tia was heated
'native' molecules ( D / N ) and T is the absolute to an elevated temperature and the absorbance
temperature in degrees Kelvin. The concentration change at that temperature was recorded. The
of denatured molecules at a particular temperature solution was then cooled rapidly to the reference

T A B L E II
THERMODYNAMIC PARAMETERS FOR THERMAL DENATURATION O F S O Y B E A N T R Y P S I N I N H I B I T O R (SBTI)

L o w - t e m p e r a t u r e transition H i g h - t e m p e r a t u r e transition

AH AS AHapp S.Sapp
(kcal/mol) (cal/mol per K) (kcal/mol) (cal/mol per K)

SBTI-Ti a 13 -+ 3 37-+ I 0 97--+24 288-+71

SBTI-Ti b 13-+2 3 6 -+ 4 92-+ 8 275-+25
SBTI-Ti c 11 -+4 31 -+ 12 122--+27 365-+78
 241

I ' I ' I I
'
which has been defended in detail by Privalov [16],
among others. There is no doubt that the high-
0.14
temperature transition can be studied indepen-
0.12
dently of the low-temperature transition either by
LU
c.O
the procedure described in connection with Fig. 5
Z
< 0.10
or by picking a wavelength at which the ab-
-r-
sorbance is unaltered by the low-temperature tran-
Ld
0.08 sition (see Ref. 17). The magnitude of the ab-
Z
< sorbance changes observed during the low-
GO

5~ 0.06 temperature transition and the enthalpy associated

t,n with the change suggest, however, that a signifi-
0.04 cant change in the structure of the molecule has
taken place during the low-temperature transition.
0.02
Acknowledgement
| i

30 40 50 60. 70 80
TEMPERATURE(°C)
Fig. 5. Reversibility (O O) of thermal denaturation of
Ti a. Upper curve (O O) shows change in absorbance
after heating to the indicated temperature. Lower curve
([] []) shows absorbance change after cooling to 20°C
from the temperature indicated on the abscissa.
temperature and the absorbance change was again
noted after waiting 5 min. This cycle of heating
and cooling was repeated several times, each time
using a temperature 5°C higher than that used in
the previous heating cycle. The results of this
experiment are shown in Fig. 5. The upper curve in
this figure shows the absorbance changes observed
at the highest temperature in the heat/cool cycle.
The lower curve shows the absorbance changes
observed after cooling the sample back down to
20°C. The lower curve represents, therefore, the
expected curve for the thermal denaturation of Ti"
if no readily reversible events occurred during
heating. Clearly, the low-temperature transition
observed in the Van't Hoff plots corresponds to an
event which is readily reversed by lowering the
temperature. We have made no attempt to ex-
amine in detail the apparent irreversibility of the
high-temperature transition. It is clearly, however,
not a readily reversible transition. The question of
whether or not the readily reversible low-
temperature transition can properly be referred to
as a denaturation event is of central importance to
the so-called 'two-state' hypothesis of denaturation
This research was supported by the College of
Agricultural and Life Sciences, University of
Wisconsin, Madison.
References
1 Singh, L., Wilson, C.M. and Hadley, H.H. (1969) Crop Sci.
9, 489-491
2 Hymowitz, T. and Hadley, H.H. (1972) Crop Sci. 12, 197-
198
3 Hymowitz, T. (1973) Crop Sci. 13,420-421
4 0 r f , J.H. and Hymowitz, T. (1977) Crop Sci. 17, 811-813
5 0 r f , J.H. and Hymowitz, T. (1979) Crop Sci. 19, 107-109
6 Freed, R.C. and Ryan, D.S. (1978) J. Food Sci. 43, 1316-
1319
7 Hymowitz, T., Orf, J.H., Kaizuma, N. and Skorupska, H.
(1978) Soybean Genet. Newsl. 5, 19-22
8 Freed, R.C. and Ryan, D.S. (1980) Biochim. Biophys. Acta
624, 562-572
9 Leach, B.S. and Fish, W.W. (1977) J. Biol. Chem. 252,
5239-5243
10 Edelhoch, H. and Steiner, R.F. (1963) J. Biol. Chem. 238,
931-938
I I Wu, Y.V. and Scheraga, H.A. (1962) Biochemistry 1,905-
911
12 Sweet, R.M., Wright, H.T., Janin, J., Chotina, Ch.H. and
Blow, D.M. (1974) Biochemistry 13, 4212-4228
13 Zahnley, J.C. and Davis, J.G. (1970) Biochemistry 9, 1428-
1433
14 Walsh, K.A. (1970) Methods Enzymol. 19, 41-63
15 Donovan, J.W. and Beardslee, R.A. (1975) J. Biol. Chem.
250, 1966-1971
16 Privalov, P.L (1979) in Advances in Protein Chemistry
(Anfinsen, C.B., Edsal, J.T. and Richards, F.M., eds.), Vol.
33, pp. 167-24t, Academic Press, New York
17 Khechinashvili, N.N., Privalov, P.L. and Tiktopulo, E.I.
(1973) FEBS Lett. 30, 57-60