In previous work on the denaturation of antibody, studies have been made of some of the factors influencing the inactivation of diphtheria antitoxin in urea solutions (1, 2). A quantitative formulation of a simple kinetic theory was found to fit satisfactorily the experimental data and to offer a reasonable explanation of the deviation of the inactivation from simple first order behavior (2). In the present work we have studied the inactivation of Staphylococcus antitoxin, investigating the influence of certain new factors on the rate and course of the over-all reaction, with a view toward gaining further insight into the mechanism of the reactions, and reinvestigating the influence of pH, a factor studied previously with diphtheria antitoxin, in order to test the applicability of the proposed kinetic mechanism to different antibodies

Schomaker, Verner

Wright, George G.

English

Caltech Authors - Main

STUDIES ON THE DENATURATION OF ANTIBODY 
IV. THE INFLUENCE OF pH AND CERTAIN OTHER FACTORS ON THE 
RATE OF INACTIV$TION OF STAPHYLOCOCCUS ANTITOXIN 
IN UREA SOLUTIONS 
BY GEORGE G. WRIGHT AND VERNER SCHOMAKER 
(Prom the Gates and Crellin Laboratories of Chemistry, California Institute of 
Technology, Pasadena)* 
(Received for publication, January 24, 1948) 
In previous work on the denaturation of antibody, studies have been 
made of some of the factors influencing the inactivation of diphtheria anti- 
toxin in urea solutions (1, 2). A quantitative formulation of a simple 
kinetic theory was found to fit satisfactorily the experimental data and to 
offer a reasonable explanation of the deviation of the inactivation from 
simple first order behavior (2). In the present work we have studied the 
inactivation of Staphylococcus antitoxin, investigating the influence of cer- 
tain new factors on the rate and course of the over-all reaction, with a view 
toward gaining further insight into the mechanism of the reactions, and re- 
investigating the influence of pH, a factor studied previously wit,h diph- 
theria antitoxin, in order to test the applicability of the proposed kinetic 
mechanism to different antibodies. 
Materials and Methods 
The antitoxic globulin was obtained from crude horse plasma’ by frac- 
tionation between 1.33 and 1.68 M ammonium sulfate. The final prepara- 
tion contained about 9 per cent protein and 269 I.U. of antitoxin per ml. 
Only the neutralizing power for the Xtaphylococcus a-toxin, which hemolyzes 
rabbit red blood cells, was considered in the present work. The hemolytic 
toxin was obtained from a semisolid agar culture of Xtaphylococcus aureus 
in the usual manner; the toxin was preserved with merthiolate and used 
in its crude form. The antitoxin assays were carried out in the customary 
manner as described previously (3). The procedure consists essentially of 
determining the dilutions of the unknown and of the treated antitoxins re- 
quired to reduce the toxicity of a constant amount of the toxin, containing 
about 150 minimal hemolytic units, to a point where only 50 per cent hemol- 
ysis of added rabbit red blood cells occurs after standard incubation. The 
ratio of the dilution of the partially inactivated antitoxin to the dilution of 
* Contribution No. 1129. 
*Supplied by the Lederle Laboratories Division, American Cyanamid Company, 
Pearl River, New York. 
169 
170 DEXATURATIOX OF ASTIUODT. IV 
the untreated antitoxin gives the proportion of antibody activity remaining; 
micro-Kjeldahl determinations (4) were necessary to correct for the random 
volume changes which occurred during the dialysis of the samples for the 
removal of the urea. The over-all determination appears to be accurate to 
within a few per cent. 
RESULTS AND DISCUSSION 
InjiTuence of pH on Rate of Inactivation-In the previous work on the 
kinetics of the inactivation of diphtheria antitoxin in urea it was observed 
that the pH markedly influenced the course of the reaction. Kinetic ex- 
periments were performed with the Staphylococcus antitoxin at a comparable 
series of pH values and in a similar manner. 8 M urea and a protein con- 
lo 
-iIM%N If&S 
40 20 40 60 SO DO 
TIME IN HOURS 
FIQ. 1 FIG. 2 
FIGS. 1 AND 2. Per cent antibody remaining (logarithmic scale) as a function of 
time of denaturation in 8 M urea at 25”, studied at various pH values. Fig. 1, 0 pH 
4.97, 0 pH 5.38, 0 pH 6.21; Fig. 2, 0 pH 7.40, 0 pH 8.92, 0 pH 9.34, 0 pH 931. The 
curves represent the equation [T] = T1e-klt + TzeMX2t. For definitions of symbols 
see Wright and Schomaker (2). 
centration of 0.3 per cent were used; the denaturation reactions were carried 
out in a thermostat at 25’. The buffer salts were held at an ionic strength 
of 0.1. Borate buffer was used at pH 9.81, 9.34, and 8.92, phosphate at 
pH 7.40, and acetate at pH 6.21, 5.38, and 4.97. Samples were removed at 
the required intervals, diluted with an equal volume of saline, and dialyzed 
in the cold to remove urea, and the per cent of the original antibody activity 
was then measured. The experimental results are given in Figs. 1 and 2. 
It is clear that the general features of the influence of pH on the inactiva- 
tion of Staphylococcus antitoxin and diphtheria antitoxin are closely similar. 
Application to the data of the theoretical treatment proposed previously for 
the inactivation of diphtheria antitoxin in urea solutions gave the curves 
presented in Figs. 1 and 2; the rate constants derived from the theoret,ical 
G. G. WRIGHT AND V. SCHOMAKER 171 
curves are given in Table I. This treatment and the proposed kinetic 
mechanism of which it is the quantitat’ive rspression have been developed 
and discussed in detail previously (2). Briefly, however, the proposed 
mechanism may be represented by t,he diagram 
where N represents the native, undenatured antibody, I the inactivated 
antibody, P a hypothetical “protected antibody” which is fully active (or 
becomes so on removal of urea) but is not susceptible to inactivation, and 
kl, kz, and 1~~ represent first order rate constants for the reactions as indi- 
cated. Examination of Figs. 1 and 2 indicates that the present data are 
well represented by this treatment, since the experimental points are, in 
general, closely fitted by the curves. The values of the rate constants de- 
TABLE I 
Variation with pH of Rate Constants Obtained from Quantitative Treatment Described 
in Text 
___- 
PH 
4.97 
5.38 
6.21 
7.40 
8.92 
9.34 
9.81 
.-_____- -. -~- 
-- 
hrr.-1 
2.54 
0.83 
0.64 
0.46 
0.24 
0.30 
1.31 
- 
RI 
__- 
hrr.-’ 
2.04 
0.94 
1.62 
1.02 
0.30 
0.41 
1.49 
- 
i 
hrs.-1 
0.0023 
0.0020 
0.013 
0.0049 
0.013 
0.035 
0.10 
rived from the theoretical curves and given in Table I, moreover, appear to 
be identical with those obtained in the experiments with diphtheria anti- 
toxin (2), within the rather large probable errors associated wit,h this sort of 
curve fitting when the number of experimental points is small. 
The satisfactory fit of the experimental data to the theoretical treatment 
outlined above appears to be additional evidence of the validity of this ex- 
planation. If the proposed kinetic explanation is in fact substantially 
correct, the striking similarity of the values of the calculated rate constants 
for the two antitoxins suggests that corresponding regions of the protein 
molecules, with essentially identical major stabilizing bonds, may be in- 
volved in the structures of the combining groups of the antibodies for differ- 
ent antigens. 
Influence of Neutral Salts on Rute of hactivatkm-An interesting aspect 
of the densturing action of urea and related substances is the influence of 
electrolytes on the reaction. The denaturing activity of guanidine salts 
172 DENATURATION OF ANTIBODY. IV 
was observed to be markedly influenced by the nature of the anion (5). 
Burk (6) studied the effect of a large number of salts on the concentration 
of urea necessary for the liberation of sulfhydryl groups of several proteins, 
and observed very striking differences, the effects varying from strong 
inhibition of denaturation to striking augmentation. The rate of denatura- 
tion of tobacco mosaic virus in urea was also observed to be influenced by 
the kind and quantity of electrolyte in the solution (7). 
Experiments were carried out to determine whether the inactivation of 
antitoxin is similarly influenced by salts. It was considered possible that 
the different component reactions of the over-all inactivation might be in- 
fluenced in different ways, a result which might lead to a separation of the 
constituent reactions and a better understanding of their nature. Three 
lo 
TIME IN HOURS 
FIG. 3. Per cent ant’ibody remaining (logarithmic scale) as a function of time of 
denaturation in 6 M urea at 25”, in the presence of 0.25 M concentrations of various 
salts, as follows: 0 none, 0 sodium sulfate, @ sodium chloride, 0 calcium chloride. 
The curves are empirical. 
representative salts were selected from t’&e large group studied by Burk (6). 
Calcium chloride was chosen as a denaturation-favoring salt, sodium sulfate 
as a denaturation-inhibiting salt, and sodium chloride as an intermediate 
salt. The urea concentration was 6 M. Because of the large influence of 
pH on the rate and course of the over-all reaction, it was necessary to control 
this factor by the addition of acetate buffer at a final concentration of 0.05 
M. The experiments were set up in the usual manner, the different salt 
solutions being added to measured portions of a solution containing the urea 
and the buffer, to give a final concentration of 0.25 M. After the solutions 
had reached temperature equilibrium in the 25” thermostat, the experiment 
was started by adding the antibody globulin. The samples were removed 
at intervals, immediately dialyzed, and assayed for antibody activity as 
before. The results are given in Fig. 3. 
G. G. WRIGHT AND V. SCBOMAKER 173 
Some variation in pH did occur in the experiment, since the pH readings 
of the solutions were as follows: control 5X%, calcium chloride 5.33, sodium 
sulfate 5.54, sodium chloride 5.52. These differences appear to bc consid- 
erably too small to account for the observed effects on the rate of inactiva- 
tion, however. 
It is clear that the influence of the three salts on the inactivation of anti- 
body parallels their influence on the liberation of sulfhydryl groups in other 
prot,eins. Thus the addition of calcium chloride promoted the inactivation, 
sodium sulfate inhibited it, and sodium chloride was intermediate in its 
effect. Them is, however, a slight difference in the behavior of sodium 
chloride, since it promoted the inactivation slightly, whereas it slightly 
inhibited the liberation of sulfhydryl groups (6). Thus the results provide 
additional evidence of the general similarity of the inactivation of antibody 
to other reactions of proteins which occur in urea solutions and which have 
been considered classical manifestations of protein denaturation. Although 
the data are not sufficiently extensive to permit the application of the quan- 
titative treatment of the reaction discussed above, it is clear that a very 
considerable change in the values of the rate constants in this treatment 
would be required to produce the large differences in initial slopes among 
the different experiments. Thus the effects of the salts on the reaction 
rates may be considerably greater than would appear from a casual inspec- 
tion of the data for the over-all reaction. It seems as though the three rate 
constants are affected by the salts in approximately the same manner. 
InJEuence of Hydrostatic Pressure on Rateof Inactivation-It has frequently 
been observed that the rate of denaturation of proteins is decreased by a 
moderate increase in hydrostatic pressure, indicating that there is an appre- 
ciable volume increase when a protein molecule goes from the normal to the 
activated state for this reaction. Studies of this sort on the denaturation 
of the bacterial luminescent system have been particularly complete (8), 
but evidence has accumulated that denaturation of many other proteins is 
also accompanied by an increase in volume (9). The rate of denaturation 
of Staphylococcus antitoxin at 65” in dilute salt solutions was observed to 
decrease with increasing hydrostatic pressure, thus providing additional 
evidence of the close relationship of antibody inactivation and protein 
denaturation (3). The influence of hydrostatic pressure on the denatura- 
tion of proteins in urea solutions does not appear to have been investigated. 
An experiment was performed to determine the influence of hydrostatic 
pressure on the inactivation of the antitoxin in 6 M urea solutions at 25”. 
Sodium acetate-sodium chloride buffer Its adjusted to give an ionic 
strength of 0.1 and pH 5.57. As soon as the globulin was added, four 5 ml. 
samples of the solution were placed in tubes, stoppered with rubber stoppers 
to leave no air space, placed in the pressure chamber, and a pressure of 
10,000 pounds per sq. in. applied. The pressure chamber and the remain- 
174 DENATURATION OF ANTIBODY. IV 
der of the solution at atmospheric pressure were placed in the 25” Lhermo- 
stat. Samples were taken at intervals from both the atmospheric pressure 
and high pressure portions, dialyzed, and assayed as before. The results 
are given in Fig. 4. 
Within the experimental error of the method there appears to be no influ- 
ence of hydrostatic pressure on the rate of the inactivation. In view of the 
considerable influence of pressure on the rate of inactivation at 65” in the 
absence of urea, this result was rather unexpected. It seems possible, how- 
ever, that a reasonable explanation of this difference in behavior may be 
proposed. The action of urea is generally believed to involve the weakening 
of the numerous secondary bonds which hold the polypeptide chain in its 
native configuration (10). High temperature, on the other hand, pre- 
sumably does not weaken the intramolecular bonds significantly, but breaks 
FIG. 4. Per cent antibody remaining (logarithmic scale) as a function of time of 
denaturation in 6 M urea at 25” at atmospheric pressure (0.) and at 10,000 pounds per 
sq. in. (0). The curve is empirical. 
them by means of locally accumulated kinetic energy. The activated state 
for this latter process might well involve a general volume increase of the 
protein, whereas the urea-saturated protein, its stabilizing bonds largely 
neutralized, could unfold without appreciable volume change. 
Rate of Change of Viscosity at Various pH ‘Values--The increase in vis- 
cosity of serum proteins when subjected to the denaturing action of urea 
is well known (11). The rate at which this increase occurs, however, does 
not seem to have been investigated to any considerable ext.ent, although 
recently the viscosity change has been observed to consist of an initial rapid 
rise followed by a gradual increase (12). Experiments were undertaken to 
determine whether the rate of the change in viscosity of the antibody 
globulin in urea could be correlated in any way with the kinetics of the anti- 
b&ly inactivation. 
G. G. WRIGHT AND V. RCHOMAKER 175 
Solutions *t a series of pH values in 8 M urea were studied. These were 
prepared in the same way as in the antibody inact,ivation experiments. 
The antibody globulin was added last, after which the solutions were quickly 
filtered to remove any trace of lint, and samples were accurately measured 
into Ostwald viseometers in a thermost’at at 25” f 0.01”. Measurements 
of the flow time were made frequent,ly at first and then at increasing inter- 
vals as the viscosity change became slower. Between measurements the 
viscometers were stoppered to prevent evaporation of the solutions. After 
completion of each experiment the viscometer was cleaned and dried and 
the flow time of the solvent was determined on a solution made up in the 
same way as the denaturation mixture (usually an aliquot of the same solu- 
tion) except that an equal volume of water was substituted for the antibody 
4 8 12 
TItlE IKOHO”~~ 
28 32 50 70 90 
FIG. 5. The relative viscosity of serum globulin as a function of time of denatura- 
tion in 8 M urea at 25’, studied at various pH values as indicated. The curvesare 
empirical. 
globulin. The relative viscosity was calculated from the two flow times, 
since density difference between t,he two solutions was assumed to be negli- 
gible. The results of five experiments at a range of pH values are given in 
Fig. 5. The early points in each experiment represent single measurements 
of the time of flow, but later measurement.s are the average of several 
measurements. 
It is clear that the behavior of the viscosity of the solutions varied greatly 
with the pH and that the results show certain similarities to those of the 
antibody inactivation experiments. In acid solution the viscosity reached 
essentially its final value rapidly. At pH 6.30 the initial change was slower 
but did seem to approach a limiting value. At pH 8.82 the viscosity con- 
tinued to increase for the duration of the experiment. At pH 9.59 a similar 
behavior was noted until about 20 hours, when the viscosity leveled off and 
176 DENATURATION OF ANTIBODY. IV 
finally began t,o decrease. This suggests that at this high pH some reac- 
tior other than the denaturation under consideration, for example a hydrol- 
ysis of the peptide bonds, contributed to the change of viscosity. Thus 
the results are similar to those obtained in the antibody inactivation experi- 
ments in certain respects and cert’ainly confirm t,he existence of relatively 
slow changes in the protein when exposed to the denaturing action of urea. 
An adequate test of the applicability to the viscosity changes of the theory 
proposed for the antibody inactivation (2) would be very difficult to make, 
however, because it would be necessary to know the viscosities of the differ- 
ent molecular species in the various buffer-urea solutions and to have infor- 
mation about the manner in which their contributions add to produce the 
observed viscosity. 
SUMMARY 
A study has been made of the influence of pH, of certain neutral electro- 
lytes, and of hydrostatic pressure on the rate of inactivation of equine 
Staphylococcus antitoxin in urea solutions and of the viscosity changes which 
take place during the denaturation. Deviations from simple first order 
behavior similar to those encountered in the inactivation of diphtheria anti- 
toxin were observed, and the course of the react.ion could be similarly 
explained by the simple kinetic mechanism proposed previously, involving 
two competing reactions of the native protein. The influence of certain 
electrolytes on the inactivation of the antitoxin was observed to be generally 
similar to their influence on the liberation by urea of sulfhydryl groups in 
other proteins. Hydrostatic pressure was found to have little or no effect 
on the rate or course of the reaction. The rate of the viscosity changes of 
the protein in the urea solution was also observed to vary with the pH, in 
a manner which was suggestive of the variation of the inactivation reaction, 
and confirmed the occurrence of relat,ively slow changes of the protein in the 
urea solution. The meaning of the results was discussed. 
This investigation was carried out with the aid of a grant from the Rocke- 
feller Foundation. The authors are indebted to Professor Linus Pauling 
for helpful discussion and to Miss Phyllis Carpenter for technical assistance 
during part of the work. 
BlBLIOGRAPHY 
1. Wright, G. G., J. Exp. Med., 79,455 (1944) ; 81,647 (1945). 
2. Wright, G. G., and Schomaker, V., J. Am. Chem. Sot., 70, 356 (1948). 
3. Johnson, F. H., and Wright, G. G., Proc. Nat. Acad. SC., 32, 21 (1946). 
4. Koch, 1’. C., and M&&kin, T. L., J. iln/. Chew). Sot., 46, 2066 (1924). 
G. G. WRIGHT AND V. SCHOMAKER 177 
5. Greenstein, J. P., J. Rid. Chem., 125, 501 (1938); 128, 233 (1939); 130, 519 (1939); 
136, 795 (1940). 
6. Burk, N. F., J. Phys. Chem., 47, 104 (1943). 
7. Lauffer, M. A., J. Am. Chem. SW., 65, 1793 (1943). 
8. Johnson, F. H., Eyring, H., Steblay, It., Chaplin, H., Huher, C., and Gherardi, 
G., J. Gen. Physiol., 28, 463 (1945). 
9. Eyring, H., .Johnson, F. H., and Gensler, R. L., J. Phys. Chem., 50, 453 (1946). 
10. Mirsky, A. IX., and Pauling, L.,-Proc. Nat. Acad. SC., 22, 439 (1936). 
11. Anson, M. I,., and Mirsky, A. E., J. Gen. Physiol., 15, 341 (1932). 
12. Royer, P. D., Ballou, G. A., and Luck, J. M., J. Biol. Chem., 162, 199 (1946). 


Studies on the denaturation of antibody. IV. The influence of pH and certain other factors on the rate of inactivation of Staphylococcus antitoxin in urea solutions

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Studies on the denaturation of antibody. IV. The influence of pH and certain other factors on the rate of inactivation of Staphylococcus antitoxin in urea solutions

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