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					     THE DENATURATION OF STAPHYLOCOCCAL
                  PROTEINS'
                  A. P. KRUEGER Aim V. C. NICHOLS
   Department of Bacteriology, University of California, Berkeley, California
                    Received for publication, May 20, 1935
   Bacteriologists employ the term "denaturation" in a loose sense
to imply alteration of antigenic complexes without defining clearly
the nature or extent of these changes. It is questionable whether
such use of the term is justified, for from the biochemists' view-
point "denaturation" has a specific chemical connotation and
refers to an intramolecular rearrangement of the protein molecule
evidenced by certain altered physical and chemical properties
which in turn may lead to gross changes in state. Proteins in
general become insoluble at the iso-electric point when de-
natured (Hardy, 1899); they lose their capacity for crystalliza-
tion; their solutions show a large increase in viscosity (Anson
and Mirsky, 1932), a rise in specific optical rotation (Young,
1922) and sulphydryl or disulphide groups undetectable in the
native protein can be demonstrated by means of color tests and
oxidation-reduction reactions (Arnold, 1910; Harris, 1923;
Walker, 1925). As compared to other chemical reactions, pro-
tein denaturation has a characteristically high temperature co-
efficient, a rise of 10'C. increasing the rate of reaction several
hundred times (Chick and Martin, 1910, 1911). This character-
istic offers a basis for inferring that such processes as the inactiva-
tion of enzymes and the killing of bacteria by heat involve basi-
cally a protein denaturation since they too possess extraordinarily
high Qlos. Proof of this hypothesis, more satisfactory than
analogy, is available in the case of pepsin, for when it is heated
the loss of activity is paralleled quantitatively by its denatura-
tion (Northrop 1930).
   Supported by Grant-In-Aid of Research from Eli Lilly and Company.
                                      401
402            A. P.   KRUEGER AND V. C. NICHOLS

   In an earlier publication one of us outlined a method for the
preparation of bacterial antigens (Krueger, 1933) designed par-
ticularly to avoid denaturation of the cellular proteins. Briefly
the technic consisted of the following steps: 1. Growth of mass
cultures. 2. Washing of organism in Locke's solution to remove
metabolities. 3. Fragmentation of cells in a special ball mill.
4. Filtration of the ground suspension through acetic-collodion
ultrafilters. 5. Standardization of the sterile filtrate on the basis
of native protein content.
   Clinical experience with such undenatured antigens has in-
dicated that they have a considerable range of therapeutic and
prophylactic application (Frawley, Stallings and Nichols, 1934;
Frawley, 1934; Stallings and Nichols, 1934; Munns and Aldrich,
1934; Kracaw, 1934; also unpublished work by Stallings and
Nichols, Kracaw and Hosmer) and that their use is attended by
a minimum of non-specific reactions on the part of the patient.
While our preparations contain known concentrations of native
proteins we were hampered by lack of specific information as to
their properties and could not state except in a very general way
just how the antigens should be handled to avoid denaturation.
Of particular practical importance was the question of tempera-
ture effects, not only for defining the limits within which the
proteins could be maintained in the native form but also for
demonstrating experimentally how an antigen made by this special
technic differs from a heat-killed vaccine. The present paper is
a record of experimental work on the heat denaturation of
staphylococcal proteins.
                          EXPERIMENTAL
   Fairly concentrated preparations of UBA (undenatured bac-
terial antigens) were made according to the method of Krueger
(1933) using several strains of Staphylococcus aureus. They all
gave the usual qualitative tests for protein and strongly positive
Molisch tests. Direct determinations were made of total protein
nitrogen and the nitrogen associated with the component native
and denatured protein fractions. In all cases the denatured
protein fraction was between 2 and 10 per cent of the total pro-
                 DENATURATION OF STAPHYLOCOCCAL PROTEINS                      403
tein present. The solutions were exposed to various tempera-
tures in a constant temperature bath; samples were taken at
intervals and the denatured protein nitrogen, the native protein
nitrogen and the non-protein nitrogen content determined as
outlined under "methods." In this way it was possible to follow
the course of the denaturation reaction in protein preparations
representing the naturally occurring compounds of the bacterium,
unaltered except as they may have been modified by rupture of
the cell membrane and mechanical agitation.

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        -~       HEAT   DEN4ATUJOATION   OF   STAPHRYLOCOCCAL PR TEONS


                                                   HO0UR35
          FIG. 1. HEAT DENATURATION OF STAPHYLOCOCCAL PROTEINS
   Total nitrogen content = 41.5 mgm. per 100 cc. Total protein content 5
89.0 mgm. per 100 cc. Denatured protein content = 7.55 mgm. per 100 cc. Na-
tive protein content = 81.45 mgm. per 100 cc.

   In figure 1 are shown the denaturation curves for 400, 450, 500,
and 550C. on one particular preparation made from a single strain
of staphylococcus known as S-3-k, plotted as logarithms of resid-
ual native protein in milligrams per 100 ml. against time of
exposure. For each temperature there was apparently a primary
rapid denaturation reaction followed by a second slower denatura-
tion. Considering only the first or rapid phase of denaturation
at each temperature the curves follow the course of a monomolec-
404                  A. P.   KRUEGER AND V. C. NICHOLS

ular reaction. The velocity constants calculated from k =
1
  In PO for these portions of the curves are:
t PO-PD
              40C.                   456C.                500C.          55C.
      k   =   0.00806        k   =   0.0181         k    =   0.064   k   =   0.198

where: k = velocity constant
         t = time of exposure
         PO = original native protein per millimeter
         PD = protein denatured in time (t).
  The van't Hoff-Arrhenius equation for the relationship between
rate of reaction and the absolute temperature states that:

                                        k/=
                                          2ie   (       T2Ti /

where: k2 = velocity constant at T2
         ki = velocity constant at T1
         T2 = second absolute temperature
         T, = first absolute temperature
         ,u = critical thermal increment.
  As a test for the constancy of ,s over the temperature range
studied the logarithms of the velocity constants are plotted
against reciprocals of the absolute temperature in figure 2.
Apparently j. for the heat denaturation of staphylococcal pro-
teins is constant within the temperature range 40 to 550C. and
has a value of about 44,000.
  In addition to the development of insolubility at the iso-electric
point another property distinguishing denatured proteins is the
appearance of sulphydryl groups. Our solutions of staphylococcal
proteins as originally prepared gave no color reaction for SH
groups, but after heating the nitroprusside reaction of Harris
(1923) became strongly positive. In figure 3 the logarithms of
sulphur in the SH form expressed as mg. of sulphur per milliliter
are plotted against the time of exposure to heat; the SH groups
appear rapidly as the protein solution is heated.
   Other preparations of staphylococcal proteins from various
strains of Staphylococci have given results, in general, compar-
able to those noted above for one particular preparation.
         -0.5

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                   OVER R 4NGE      40e     -  55'C

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            3.c3                          3.1                       3.15              3.2        3.25
                                            1/T
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FIG. 2. GRAPHIC TEST FOR CONSTANCY OF CRITICAL THERMAL INCREMENT OVER
                      TEMPERATURE RANGE STUDIED




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                                   DEVELOPME iT OF SULPHYD ZYL G
                                                               GQOUDS
                                           31
                                   DUQING MAT DENATUR .TION OF    O
                                   STAPHYLO OCCAL PRO EIN5.     .

                           -2.0
                                           0.25                         0.5           0.75
                                                   HOURS
FIG. 3.    TITRATION       OF   SH GROUPS DURING HEAT DENATURATION                           OF STAPHY-
                                     LOCOCCAL PROTEINS
                                            405
406           A. P.   KRUEGER AND V. C. NICHOLS

                            METHODS
   1. Nitrogen determinations were carried out by the micro-
Kjeldahl method as described by Pregl (1930) using the apparatus
of Parnas and Wagner (1921).
   2. For each preparation of undenatured antigen the following
determinations were made:
   a. Total protein nitrogen. To 5.0 ml. of solution was added
5.0 ml. of 10 per cent trichloracetic acid. The solution was kept
at 200C. one-half hour, centrifuged at high speed for fifteen
minutes and the nitrogen content of the precipitate determined.
Throughout our work it was,necessary to use special digestion
flasks in which the small amounts of precipitate could be packed
solidly; loss of materials was avoided in this way.
   b. Total non-protein nitrogen. A direct Kjeldahl determination
was run on 5.0 ml. of the supernatant from (a).
   c. Denatured protein. One milliliter of citrate buffer (S0ren-
sen's citrate-HCl mixture) pH 4.6 was added to 4.0 ml. of solution
and the mixture was kept at 200C. for ten minutes. After cen-
trifugation at high speed the supernatant was poured off into
another digestion flask for (d) and a determination run on the
precipitate. This latter figure gave the nitrogen equivalent of
the denatured protein.
   d. Native protein determination. To the supernatant from (c)
there was added 5.0 ml. of 10 per cent trichloracetic acid. The
mixture was kept one-half hour at 200C. and was then centrifuged.
A Kjeldahl determination was made on the precipitate giving the
native protein nitrogen content.
  3. To follow the course of the denaturation reaction 4.0 ml.
aliquots of antigen were placed in the special digestion tubes and
exposed to 400, 450, 500, and 550C. in the water bath. At
intervals tubes were removed and 1.0 ml. of citrate buffer pH 4.6
added to the antigen. The tubes were held at room temperature
ten minutes and then centrifuged. The supernatants were
poured off into another digestion tube and a Kjeldahl determina-
tion was made on the precipitate. This gave a direct measure of
denatured protein. To 5.0 ml. of supernatant there was added
5.0 ml. of 10 per cent trichloracetic acid. After centrifugation a
determination was run on the precipitate and on the supernatant
         DENATURATION OF STAPHYLOCOCCAL PROTEINS                407
giving respectively the residual native protein nitrogen and the
non-protein nitrogen.
  With the above data available for each preparation it was
possible to check back and to determine the percentage error at
each step and also to ascertain whether protein hydrolysis was
occurring. In the experiments cited the loss of protein by hy-
drolytic cleavage was extremely small.
  4. For the quantitative determination of SH groups standard
iodine in KI was added to aliquots of the test solution and the
residual iodine remaining after reaction with available SH groups
was determined by back titration with standard sodium thio-
sulfate solution. The general equation for the reaction is:
              2 RSH+ I2 -R - S - S - R+2HI
                            DISCUSSION
   The experimental data on the course of the heat denaturation
of staphylococcal proteins indicate that there is first a rapid dena-
turation followed by a slower denaturation for each of the tem-
peratures studied. The reactions proceed logarithmically with
time and have a critical thermal increment of the same order of
magnitude as the values reported for the spontaneous destruction
of a variety of proteins. For example, Chick and Martin (1910,
1911) studied the heat denaturation of hemoglobin in distilled
water over the range 600 to 70'C. and found the temperature co-
efficient to be 13 corresponding to a j, of about 55,000. Our
preparations of staphylococcal proteins gave an average of A =
44,000. This difference in magnitude of p is not serious and may
be attributed to the natural variation between different proteins
and the effects of the conditions under which the experiments are
performed. Denaturation as a rule proceeds most rapidly at the
iso-electric point (Chick and Martin, 1910, 1911) and is inhibited
by the presence of salts (Handovsky, 1910). The conditions
obtaining in our work were not optimal, for the pH was 7.2 (iso-
electric point = 4.6) and the solvent employed was Locke's
solution. We found that runs made with solutions adjusted to
pH 4.6 showed material increases in velocity, u increasing as high
as 53,000.
408            A. P.   KRUEGER AND V. C. NICHOLS

   If the value 1A = 44,000 applies to the additional 50 increment
between 550 and 60'C. as it does to the 400 to 550C. range for
which it has been shown to hold, then k = 0.54 at 600C. Using
this velocity constant in the equation for the velocity of a mono-
molecular reaction to calculate the amount of native protein
nitrogen left after four minutes' exposure to a temperature of
600C. it is found to be 1.61 mgm. or less than 12 per cent of the
original amount present.
   Our data then suggest that at the temperature ordinarily
employed for killing bacteria in the preparation of vaccines, i.e.,
60WC., substantially all of the staphylococcal proteins are de-
natured. It is possible to avoid this denaturation to a very con-
siderable degree by substituting mechanical fragmentation of the
cells for heat treatment as outlined in a previous publication
(Krueger, 1933). Further, it would appear that undenatured
bacterial antigens made according to this technic may be main-
tained at ordinary room temperature without danger of dena-
turation. Temperatures of 40TC. and over will produce rapid
denaturation of the constituent proteins. It is recognized that
our studies deal with rather crude solutions and, purposely, no
attempt has been made to purify them in order that our data
might be interpreted as applying to the preparations used for
clinical work.
                   SUMMARY AND CONCLUSIONS
   1. Staphylococcal antigens made by mechanical fragmentation
and ultrafiltration according to the method of Krueger, contain
less than 10 per cent of the total bacterial protein in the denatured
form.
   2. On exposure to heat, staphylococcal native proteins are
denatured, becoming insoluble at the iso-electric point (pH 4.6)
and showing a considerable increase in SH groups. The de-
naturation reaction obeys the Mass Law and has a critical ther-
mal increment averaging about 44,000.
   3. These data have practical implications in regard to the heat
killing of bacteria for vaccines and also with respect to the
conditions necessary for preservation of undenatured bacterial
antigens.
          DENATURATION OF STAPHYLOCOCCAL PROTEINS            409

                               REFERENCES
ANSON AND MIRSKY 1932 J. Gen. Physiol., 15, 341.
ARNOLD 1910 Z. Physiol. Chem., 70, 300.
CHICK AND MARTIN 1910 J. Physiol., 40, 404.
         1911 J. Physiol., 43, 1.
FRAWLEY 1934a J. Pediat., 4, 184.
FRAWLEY 1934b J. A. M. A., 103, 960.
FRAWLEY, STALLINGS AND NICHOLS 1934 J. Pediat., 4, 179.
HANDOVSKY 1910 Biochem. Ztschr., 25, 510.
HARDY 1899 J. Physiol., 24, 158.
HARRIS 1923 Proc. Roy. Soc. B., 94, 426.
KRACAW 1934 Cal. and West. Med., 40, 228.
KRUEGER 1933 J. Inf. Dis., 53, 237.
MuNws AND ALDRICH 1934 J. Pediat., 5, 590.
NORTHROP 1930 J. Gen. Physiol., 13, 739.
PARNAS AND WAGNER 1921 Biochem. Ztschr., 125, 253.
PREGL 1930 Quantitative Organic Microanalysis. Blakiston.
STALLINGS AND NICHOLS 1934 Amer. J. Dis. Child., 48, 1183.
WALKER 1925 Biochem. J., 19, 1082.
YOUNG 1922 Proc. Roy. Soc. B., 93, 15.

				
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