The Effects of Heat on Dry Proteins. I. The Kinetics of the Formation of

Department of Biochemistry, Tulane University School of Medicine,. New Orleans, Louisiana. Received June 2, 1941. Although the presence of water is an...
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F. W. BERNHART

T H E EFFECTS O F HEAT ON DRY PROTEINS.

I

THEKINETICSOF THE FORMATION OF INSOLUBLE EGGALBUMIN F. W. BERNHART’ Department of Biochemistry, Tulane Uniz’erszty School of Medicine, New Orleans, Louasiana Received June .$, 1941

Although the presence of water is an important factor in the heat coagulation of proteins (8), the rBle of water in the heat coagulation of proteins other than those in solution has received little attention. Chick and Martin (5) found that crystalline egg albumin containing 20 per cent of mater is completely soluble after being heated for 5 hr. at 120°C. They found that 22 per cent of crystalline egg albumin containing 20 per cent of water is insoluble in water after 4 hr. of heating at 130°C. Barker (1) observed that the “denaturation temperature” (the temperature at which one-half of the protein is insoluble in distilled water after 10 min. of heating) is a linear function of the relative humidity with which the protein has been in equilibrium. The temperature of denaturation of egg albumin stored over phosphorus pentoxide was found to be 162°C. This paper is a report on the kinetics of the reaction which results i n the formation of insoluble egg albumin from dry soluble egg albumin. EXPERIMENTAL

Egg albumin was prepared by the method of Cole ( 7 ) and recrystallized twice. Ammonium sulfate was removed from the final product by dialysis against distilled water, dialysis being continued for 3 days after Sessler’s test for ammonia in the dialysis water was negative. The egg albumin was dried by blowing air a t room temperature against the protein solution, which wa5 contained in the suspended dialyzing tube (T’isking sausage casing). This method of drying the albumin was found to prevent surface denaturation almost completely. The dry protein present as a glassy maw in the bottom of the sausage casing was ground in a mortar and passed through an 80-mesh sieve. The powdered egg albumin was then stored over phosphorus pentoxide. A method was developed for the determination of the fraction of egg albumin made insoluble by heating. This method prevents the occlusion of soluble by insoluble protein and minimizes the formation of surfacedenatured protein formed by stirring during the solution process. Approximately 100 mg. of the dry powdered egg albumin was spread thinly and evenly on a tared watch glass and weighed. The sample was then 1 Present address: Department of Biochemistry, The Cleveland Clinic, 93rd Street and Euclid Avenue, Cleveland, Ohio.

EFFECT OF HEAT ON DRY PROTEINS

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heated in an air thermostat (accuracy T 0.5"C.). The protein was sprinkled as evenly as possible on the surface of about 30 cc. of water contained in a 400-cc. beaker. The large amount of water surface per milligram of protein is an insurance against the formation of gummy masses and, as no solid protein leaves the surface, solution occurs without stirring. About 20 mg. of thymol was added to guard against bacterial growth, and the mixture was allowed to stand for 12-24 hr. a t 5°C. Then the protein solution was transferred to a 100-cc. volumetric flask, and the precipitate which remained was broken up with a glass rod. The precipitate was then transferred to the volumetric flask with the aid of ten to fifteen portions of water of 3-4 cc. each. The flask was then filled to the illark and allowed to stand for 12-24 hr. to insure solution of any undissolved but soluble protein. The contents of the flask were filtered through No. 575, C. S. and S. filter paper. Use of this filter paper resulted in water-clear filtrates in all determinations. The soluble protein present was estimated from duplicate Kjeldahl analyses for total nitrogen (4) in aliquot portions of the filtrate. Control determinations, following the above technique with unheated egg albumin, were used to establish the value for soluble protein present a t the beginning of an experiment. EXPERIMENTAL RESULTS

If we let t = time,

S t = soluble protein present after heating t minutes, So = soluble protein present a t the beginning of an experiment, i.e., t = 0, 1 = fraction of So insoluble a t time t, and S = fraction of So soluble a t time t ,

then

I = (So - St)/SO

s = St/So and

S+1=l ;In example of the curves obtained when the fraction of insoluble protein present (I) is plotted as a function of the length of time the protein was heated is shown in figure 1. It was found that the reaction followed the differential equation dI/dt = k * I * S where k = rate constant.

(1)

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F. W. BERNHART

Since

1-1-8 therefore -1 5 w

cU

=

kdt

1.0-

-

0

80

160

240

320 400

TIME IN MINUTES

Fro. 1. Increase in insoluble protein with time of heating. Temperature, 136°C.

TIME IN MINUTES

FIG.2. Plot of the log of the ratio of soluble to insoluble protein present against time of heating. Curve 1, Ill'C.; curve 2,123OC.; curve 3, 136'C.; curve 4,144'C.; curve 5, 156°C.; curve 6, 164'C.; curve 7, 176OC.

Integration of equation 2 gives In S / I = - k t where C = constant of integration.

+ In C

(3)

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EFFECT OF EEAT ON DRY PROTEINS

According to equation 3 a linear relationship should exist between log S / I and t. This relationship is illustrated in figure 2. The linearity

Values of k . TEXFERAlVRE

TABLE 1 verimental and calculated valw k

x

P R h C n O N OF INSOLUBLE PROTEIN

lo,

'C.

111

123

136

144

for fraction of insoluble protein

1

Experimental

Calculated

81 210 470 532 1287

o ,016

0.015 0.018 0.031 0.034 0.13

71 237 451 599

0.016 0.031 0,079 0.16

0.017 0.033

108 142 169 202 238 327

0.10 0.17 0.28 0.45 0.68 0.90

0.10 0.18 0.28 0.46 0.64 0.91

51 112 194

0.056 0.31 0.84

o.aM

minute8

1.93

4.64

21.3

32.0

0.017 0.024 0.048 0.12

0.084

0.16

'

0.30 0.86

156

164

10 20 33 52

0.062 0.26 0.72 0.95

0.062 0.27 0.72 0.98

164

270

8 10

0.028 0.076 0.080 0.33 0.82

0.045 0.076 0 ow 0.28 0.82

0.024 0.11 0.13 0.46 0.73 0.81

0.034 0.078 0.16 0.50 0.70 0.85

11

16 25 176

887

4

5 6 8 9 10

______

of the graphs was maintained up to about 85 per cent change. The best line was fitted by eye to the experimental points, and specific reaction rates

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F. W. BERNHART

were determined by multiplying the slopes of the lines by 2.303. Values of k and the comparison between experimental and calculated values are reported in table 1. The increase in the rate of formation of insoluble egg albumin which accompanies increases in temperature is shown in figure 3. The energy of activation calculated from the simple Arrhenius equation using this straight line is 33 kg.-cal. per mole.

-3.0 I 0.0021

i

'

I

I

I

a0023

t

1

0.0025

I 0.0027

vr FIG.3. Plot of increase in rate of formation of insoluble protein against increase in temperature. DISCUSSION

The coagulation of egg albumin in solution by heat is a first-order reaction (6). I n contrast, the equations representing the kinetics of the reaction described in this paper are essentially the same as those which describe autocatalytic reactions (9). However, it is difficult to see how thermal disruption of the structure of one albumin molecule could catalyze the process of neighboring molecules. KO explanation for the kinetics of the reaction will be attempted a t this time. The data of Barker (1) indicate a relationship between the heat denaturation reaction in solution and the formation of insoluble protein studied here. His data show that the temperature required for 50 per cent denaturation in 10 min. is a linear function of the relative humidity with which the powdered egg albumin has previously beer in equilibrium. The water content of powdered egg albumin is related to the relative

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humidity with which it is in equilibrium at room temperature. These contents vary from 0.74 g. of water per gram for egg albumin in equilibrium with water vapor to 0.042 g. of water per gram for egg albumin in equilibrium with a relative humidity of 15 per cent (1). Dry powdered proteins stored over phosphorus pentoxide contain water which can only be driven off by heating to teniperatures over 100°C. (3). In the experiments described here, approximately 2 per cent masimum loss in weight occurred. The activation energy calculated from the slope of the line in figure 3 (33 kg.-cal. per mole) is considerably less than the activation energy found for heat denaturation of egg albumin in solution (140 kg.-cal. per mole) ( 5 ) . However, the activation energy value obtained here is of the same order of magnitude as the value of 35 kg.-cal. per mole calculated by Bull ( 2 ) for heat denaturation of egg albumin in solution after consideration of the influence of heats of ionization upon the observed values for the activation energy of denaturation. SUMMARY

1. A description is given of the kinetics of the reaction resulting in the formation of insoluble egg albumin when dry egg albumin is heated. 2. The activation energy for this reaction is calculated to be 33 kg.-cal. per mole. REFERESCES (1) BARKER:J . Gen. Physiol. 17, 21 (1933). (2) BULL:Cold Spring Harbor Symposia Quant. Biol. 6, 1-10 (193Sj. (3) CALVERT: In Cliemislry of the .4nrino Acids and Proteins (edited by C. L. A. Schmidt), p. 216. Charles C . Thomas, Springfield, Illinois (1938). (4) CIYETT: J . Lab. CXn. Med. 17.79 (1931). (5) CHICKASD MARTIS: J . Physiol. 40, 404 (1910). : Physiol. 43, 1 (1911). (6) (-HICK .~SDU ~ R T I SJ. (7) ( ' O L E : Proc. SOC.Esptl. Biol. Sled. 30, 1162 (1933). : S a t l . Acad. Sci. L-.S.22, 430 (1936). (8) M I R ~ K .\SD I - P A ~ I . I S GProc. (9) O S T K ~ L DJ. : prakt. Chem. 136, 181 (1883).