THE OPTICAL ROTATORY POWER OF HEAT-DENATURED EGG ALBUMIN

BY H. ALBERT BARKER

(l&n the Departmmt of CAemistry, Stanford University, California)

(Received for publication, June 14, 1933)

The most obvious change in egg albumin brought about by the action of heat, strong acids and alkalies, alcohol, concentrated urea, and other reagents is the total loss of solubility in the region of the isoelectric point. Indeed, complete insolubility at the isoelectric point, especially in the presence of neutral salts, is the primary and, for a long time, was the only definition of denatured egg albumin. Such a definition is, of course, consistent with the common classification of the native proteins on the basis of their solubilities. Nevertheless, this definition is unsatisfactory be- cause it is purely qualitative and readily leads to the unjustified assumption that the products resulting from the treatment of native egg albumin with any of the above mentioned reagents are all identical provided only that they show the same insolubility.

We have therefore undertaken more adequately and quantita- tively to characterize one derivative of native egg albumin; namely, that produced by the action of heat, by means of its optical rotatory power. To this end we have studied the depend- ence of the specific optical rotation of heat-denatured egg albumin upon the time and temperature of heating, the hydrogen ion and protein concentrations of the heated solution, and the state of ionization of the protein.

Comparatively few observations and no systematic studies have been made on the optical rotation of denatured egg albumin. Nevertheless, it has long been known that in general the rotation of protein solutions is increased by those changes which are collec- tively called denaturation. The most strongly acid solution ob- served by Frisch, Pauli, and Valkb (1925 ) displayed a much greater rotatory power than the less acid solutions. Holden and Freeman

I

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2 Heat-Denatured Egg Albumin Rotation

(1930) invariably observed an increase in rotation whether acid, alcohol, or heat was used as the denaturing agent. They state that numerous unsuccessful attempts were made to prepare specimens of denatured egg albumin of definite and constant specific rota- tion. The range observed was from [ -58.8°]s461 for a sample prepared with cold alcohol to [ -99’1 6dG1 after prolonged treatment with 0.5 N hydrochloric acid at 37”. Further, they observed a slow change in rotation in acid and alkaline solutions during a period of 12 days. From these miscellaneous observations they concluded that denatured proteins might only be regarded as uncertain mix- tures which cannot be successfully characterized by their specific optical rotations.

The observations of Holden and Freeman appeared to us in- sufficient completely to justify the conclusions drawn from them. It seemed at least possible that a more systematic study of the conditions of a single kind of denaturation would show upon what the variations of rotatory power depend.

Since egg albumin solutions freed from salts by prolonged dialy- sis or even electrodialysis are coagulated by heat in the region of the isoelectric point, the rotatory power may be studied only in solutions sufficiently acid or alkaline to prevent flocculat,ion. The acid protein solutions seem the less desirable to use because, due to the position of the isoelectric point, only solut,ions so acid as to cause decomposition of the protein (Sjogren and Svedberg, 1930) suffice to hold heat-denatured egg albumin in solution. Salt- free solutions, alkaline to the isoelectric point, do not coagulate nor become opalescent above about pH 6.7. There is thus a wide zone of pH 6.7 to about pH 10.0 in which the native egg albumin at least is stable in the sense of retaining its normal molecular weight and being monodisperse (ultracentrifuge). We have there- fore chosen to study the rotatory power of the sodium salts of denatured egg albumin.

Criticism of the use of solutions alkaline to the isoelectric point might, however, be made on the basis of the work of Sorensen and Sorensen (1925). They showed that the decomposition of coagu- lated egg albumin (measured by the increase in soluble nitrogen) due to the action of boiling water is at a minimum in the isoelect,ric region and increases markedly wit.h increasing distance of the pH of the solut,ion from t,his region. However, even the total nitro-

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H. A. Barker 3

gen liberated after 3 hours boiling at pH 6.1 amounted to only 0.11 per cent of the original nitrogen present. This figure would be greatly reduced if the time and temperature of heating were greatly decreased as in our experiments. We may therefore as- sume as a first approximation that decomposition of an indefinite sort does not distort our measurements of the rotatory power of alkaline, heat-denatured egg albumin.

Methods and Materials

The st,ock solutions of egg albumin were prepared according to the method of S$rensen and Hoyrup (1917) and were either two or three times recrystallized and subsequently dialyzed or electro- dialyzed until practically free from salts. Stock Solution D-3, with which all of the experiments on opt,ical activity here recorded were carried out, possessed a specific conductivity of 6.75 X 10e6 reciprocal ohms in 1 per cent solution at 25”, a pH of 4.92, and a specific = 5461 A.

optical rotation of 37” for the mercury green line, X

The pH of the protein solutions was adjusted with COTfree sodium hydroxide. The pH measurements were made with a glass electrode, with two standard buffer solutions for reference values.

The protein concentration was determined by drying samples of the solution to constant weight at 110” and making a correc- tion for the acid or alkali added to give the desired pH. The pro- tein stock solution was assumed to be salt-free.

A Schmidt and Haensch, Lippich, half shadow polarimeter with a three section field of view, reading to O.Ol”, was used for rotation measurements. The source of light was a mercury arc provided with filters to give only the green line, X = 5461 A.

A water thermostat was used which gave temperatures constant t,o within f0.15’.

EXPERIMENTAL

Dependence of Rotatory Power upon Time and Temperature of Heating-The following procedure was used. An egg albumin solut,ion of suitable concentration and pH was prepared and di- vided among several Pyrex test-tubes. These were immersed in the water thermostat at the desired temperature and were with-

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4 Heat-Denatured Egg Albumin Rotation

TABLE I

Efeect of Time and Temperature upon Specijic Optical Rotation, [a], of Alkaline, Heat-Denatured Ena Albumin Solution D-3. 10 Cm.

Temptmture

“C.

70.0

72.5

75.0

80.0

80.0

85.0

Time of mmemion

pR befall? heating

min.

2 4 8

16 32 64

2 4 8

16 32 64 2 4 8

16 32 64 2 3 4 5

15 30

0.5C 0.75 1.0 2 5

15 2 3 5

10 20 40

7.41 (7.40)* 7.40

(7.40) 7.40

(7.40) 7.44

(7.42) 7.40

(7.42) 7.42

(7.42) (7.44) 7.45 7.37 7.45 7.44 7.45 7.36

(7.39) 7.39

(7.39)

(Xi) (7.27) (7.27) 7.25

(7.27) (7.27) 7.29 7.48 7.40 7.43 7.40 7.38 7.43

-

I ,

--

-

a

.- P., degress dc?m3+ees

3.64 -1.34 -36.8 3.77 -1.46 -38.7 3.72 -1.47 -39.5 3.76 -1.64 -43.6 3.72 -1.79 -48.1 3.73 -2.08 -55.8 3.66 -1.36 -27.2 3.67 -1.42 -38.8 3.67 -1.60 -43.6 3.67 -2.01 -54.8 3.67 -2.33 -63.5 3.68 -2.67 -72.5 3.66 -1.39 -38.0 3.68 -1.53 -41.6 3.72 -2.18 -58.6 3.66 -2.54 -69.4 3.72 -2.76 -74.2 3.70 -2.88 -77.8 3.71 -1.64 -44.2 3.74 -2.43 -65.0 3.71 -2.57 -69.3 3.70 -2.64 -71.4 3.74 -2.82 -75.4 3.71 -2.83 -76.3 3.78 -1.36 -36.0 3.76 -1.33 -35.4 3.75 -1.37 -35.6 3.76 -1.88 -50.0 3.75 -2.71 -72.3 3.70 -2.77 -74.9 3.65 -2.46 -67.4 3.70 -2.59 -70.0 3.70 -2.64 -71.4 3.68 -2.67 -72.6 3.70 -2.75 -74.3 3.71 -2.81 -75.7

- - l Parentheserr indicate values which were not directly measured.

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H. A. Barker 5

drawn successively at various time intervals and immediately cooled in ice water. Later they were brought to room tempera- ture (22’) and the optical rot&ion was measured in 10 cm. po- l&meter tubes. The solutions were analyzed directly after the readings were completed.

The experimental results are given in condensed form in Table I and are represented graphically in Fig. 1.

It is evident from Fig. 1 that the increase of optical rotation with time of heating, under the conditions of these experiments, is a regular and continuous process. The increase is at first rapid and then much slower. It seems probable that the limiting value

04 I

TIME OF HEATING IN MINUTES

FIO. 1. The effect of temperature and time of heating upon the speoific optical rotation of alkaline, heat-denatured egg albumin, Solution D-3, at pH 7.4.

for all temperatures is the same, namely about 76-77”, although the change is too slow at the lower temperatures to reach comple- tion in 1 hour. In other words, the limiting value of the optical rotation is independent of the temperature of heating.

We are now in possession of the knowledge necessary to choose a standard time and temperature of heating for the purpose of comparing the optical rotation of heatdenatured egg albumin solutions under various conditions. We have thought it desirable to make the temperature and time as low and short as possible, respectively, in order to avoid unnecessary decomposition of the

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Heat-Denatured Egg Albumin Rotation

protein due to the actiOn of hot water and still be certain that the optical rotation has closely approached its limiting value. There- fore we have chosen a standard heating time of 20 minutes in a water bath at 80”. The protein solution (in Pyrex test-tubes) will therefore only be at 80” for approximately 17 minutes. However, in this region the time-optical rotation curve is so flat that a few minutes difference in heating time will scarcely at all affect the observed rotation. Differences in the shape and thickness of the test-tubes containing the protein solutions also will not increase the uncertainty of the observations.

It is perhaps worth pointing out in this place that measurements of the type which we have carried out might well be used in studies of the kinetics of the denaturation of proteins. This method un- doubtedly has some advantages over the analytical methods heretofore used. It is much less laborious and continuous obser- vation is possible. Our own measurements are not suitable for cal- culating the velocity constants of the reaction, however, because too few experimental points have been taken, because the tempera- ture has not been sufficiently constant, and finally because the pH of the solutions haa been allowed to vary throughout the experi- ments. Due to this last cause, most probably, the curve obtained by roughly plotting the logarithm of the optical rotation against time for one temperature is not linear but alters in such a manner as to indicate a progressively decreasing velocity constant. This same phenomenon was observed by Chick and Martin (1913) in their classical experiments on the kinetics of protein denaturation.

Dependence of Rotatory Power upon pH of Heated Egg Albumin Solution-Preliminary experiments indicated that the limiting value of the optical rotation reached aa a result of heating de- pends strongly upqn the pH of the heated solution. It must be noted that there is for the present some arbitrariness in assigning a definite pH value to a heated egg albumin solution. It is neces- sary to digress briefly in order to make this situation clear.

When an egg albumin solution is denatured by heating, there occurs, in general, an alteration in hydrogen ion concentration in the sense that in acid solutions the hydrogen ion concentration decreases whereas in alkaline solutions it increases. Somewhere near the region of neutrality the hydrogen ion concentration neither increases nor decreases.

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H. A. Barker 7

Our own observations upon the change of pH accompanying the heating of egg albumin solutions are presented graphically in Fig. 2, as are also the data of S@rensen and Jiirgensen (1911), Quaglia- riello (1912), and Chick and Martin (1913).

Despite the very evident lack of precise agreement, one may note certain general similarities in the data of the various investi- gators shown in Fig. 2. There is an unmistakable maximum in the region of pH 5 to 6. On the acid side of this maximum the pH values decrease continuously and approach the abscissa asymp- totically. This trend of the curve is, of course, an inevitable con- sequence of the method of plotting the data, for the buffer of the more acid solutions is relatively much greater than that of the less

l 83 BARKER ABOUT 470 0 Alx

-1.0 I i + 52 . QUAGLlARlELLO (1912) ABOUT 0.6% A CHICK AND MARTIN (1913) ABOUT 1.0%

-e- D2 0 S$REWN AND JijRGENSEN (1911) 0.6 - 3.6 $

FIQ. 2. The relation of the pH change accompanying the heat denatura- tion of egg albumin to the pH of the unheated solution.

acid solutions. To the alkaline side of the maximum the curve also decreases in such a way as to become zero at about pH 8. Beyond this the ApH values change sign and eventually pass through a minimum in the region of pH 9 to 10. In more alk&ne solutions the curve will again approach the abscissa asymptotically for the same reason that it did so in strongly acid solutions. One should particularly note that the isoelectric point is not distin- guished by any particular modification of the curve; the maximum, minimum, and the zero point all fall elsewhere.

The factors which influence the magnitude of the pH change are not clear from the existing data. It is true that the principle pH change accompanies the progressive loss of solubility. However,

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8 Heat-Denatured Egg Albumin Rotation

no simple relation appears to exist between the two. In addi- tion to and following the primary pH change accompanying dena-

TABLE II

Dependence of SpeciJic Optical Rotation+ [a], of Alkaline, Heat-Denatured Egg Albumin, Solution D-3, tipon Idtial pH ofoSolution. 10 Cm.

Polarimeter Tubes, h = 6.@1 A.

um. 4.87-4.9:

pH before beating

6.75 7.14 7.36 7.65 7.99 8.33 8.76 8.93 9.10 6.58 6.98 7.02 7.13 7.45 7.47 7.73 8.08 8.15 8.19 8.74 8.81 8.83 8.85

2.80-2.85 6.85 7.18 7.67 8.24 8.62 9.19

-

pH before beating

-

- degree8

-4.37 -4.20 -4.19 -4.01 -3.97 -3.88 -3.81 -3.73 -3.75 -3.30 -3.19 -3.17 -3.15 -3.05 -3.01 -3.00 -2.94 -2.90 -2.88 -2.81 -2.83 -2.83 -2.85 -2.12 -2.04 -1.94 -1.89 -1.85 -1.82

degreea

-89.6 -86.0 -85.0 -82.4 -80.8 -79.0 -77.8 -76.6 -76.2 -83.5 -81.2 -80.0 -79.8 -77.5 -76.4 -75.9 -74.1 -73.5 -73.5 -71.5 -71.3 -71.3 -71.6 -74.8 -71.8 -68.5 -67.0 -65.4 -64.1

9.48 9.49 9.56 9.56 9.64 9.96

10.20 10.33 10.42 9.03 9.30 9.42 9.47 9.75 9.90

10.04 10.09 10.30 10.41 11.02 11.16 11.31 11.36 9.35 9.53 9.90

10.11 10.29 10.42

--

degrees degrees

-3.65 -74.3 -3.61 -73.8 -3.60 -73.4 -3.61 -73.8 -3.62 -73.8 -3.50 -71.8 -3.44 -70.4 -3.42 -70.1 -3.41 -69.9 -2.76 -70.0 -2.69 -68.5 -2.74 -69.1 -2.68 -68.2 -2.65 -67.3 -2.66 -66.9 -2.66 -67.1 -2.65 -67.2 -2.65 -66.2 -2.64 -67.2 -2.65 -67.5 -2.70 -68.3 -2.77 -69.8 -2.70 -68.9 -1.80 -63.8 -1.80 -63.2 -1.80 -64.3 -1.80 -64.1 -1.81 -64.2 -1.81 -64.6

-

turation, there is a slow change in pH which may probably best be accounted for by gradual decomposition of the protein under the action of hot water.

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H. A. Barker 9

For want of a better method of expressing the pH of the solution during the denaturation, we have used the initial pH. This value should be fairly satisfactory for comparing protein solutions of otherwise similar composition.

Table II gives the data relating the specific optical rotation of a heat-denatured egg albumin solution to the initial pII. The standard heating time of 20 minutes at 80’ was used. Each sec- tion of the table corresponds to experimental solutions of a differ- ent protein concentration. The data are represented graphically in Fig. 3.

2 1 u7 -35 7.0 25 8.0 a5 90 85 DO IO.5 11.0 II.5

pH BEFORE HEATING

Fro. 3. The dependence of the specific optical rotation of alkaline, heat-denatured egg albumin, Solution D-3, upon the initial pH and concen- tration of the solution. The concentration in gm. per 100 cc. of solution is for Curve I, 4.89; Curve II, 3.95; Curve III, 2.82.

It will be observed in Fig. 3 that specific rotation values cor- responding to a given protein concentration fall along a smooth curve. The rotation decreases continuously with increasing pH in the region of pH 6.6 to 9.0, at least. In solutions more alkaline than this the specific rotation passes through a minimum and in- creases slightly. Probably the curves corresponding to different protein concentrations would converge and might coincide in very alkaline solution.

Dependence of Rotatory Power upon Egg Albumin Concentration -The three curves in Fig. 3 correspond to experiments carried out with different protein concentrations. The general form of the

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10 Heat-Denatured Egg Albumin Rotation

curves is independent of the concentration, although the position of the minimum is displaced to lower pH values in solutions of lower concentration, so that the curves are not strictly parallel over the whole range studied. A point of great interest is that the specific optical rotation of a heat-denatured egg albumin solution, referred to a particular pH value of the unheated solution, is strongly dependent upon the protein concentration. This de- pendence is nearly linear in the region of pH 7.0 to 9.0. Above pH 9.0, in the region of the minima in the pH-optical rotation curves, the linear relation is lost. The following equations give the dependence of the specific optical rotation upon protein con- centration for three pH values in the region of linearity. C is gm. of protein per 100 cc. of solution.

pH 7.0 [a] = -6.76 C - 54.0 “ 8.0 [a] = -6.47 “ - 49.2 “ 9.0 [a] = -5.65 “ - 48.1

Dependence of Rotatory Power upon State of Ionization-AU of the previously described experiments which elucidate the relation between pH and the rotatory power of denatured egg albumin were carried out by heating a series of solutions of various acidities and then measuring the optical rotation. The observed effect might therefore be due either to the act of heating at a definite acidity, or to the final acidity alone, independent of the acidity during heating. In short, the acidity might be important for the kinetic process of denaturation or it might determine an equilib- rium condition.

In order to elucidate this matter, we carried out a series of ex- periments in which one single solution was heated and cooled, and then various portions were diluted and brought to various hydro- gen ion concentrations. The optical rotation was then measured.

DISCUSSION

It will be seen from Table III that the specific optical rotation is, within the experimental error, independent of the final hydro- gen ion concentration, and that its absolute value is higher than in any of our previous experiments. The constancy of specific ro- tation with varying hydrogen ion concentration under these con- ditions undoubtedly proves that the state of ionization of the pro-

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H. A. Barker 11

tein molecules does not control the variation of specific rotation with pH of the heated solution (Fig. 3). Therefore we must admit that the other alternative is true; namely, that the pH is primarily important for the kinetic process of denaturation.

The high absolute value of the specific rotation must be similarly explained. Evidently, not the final values of the pH and concen- tration, but the values during heating determine the observed rotation. We have seen that lowering the pH and increasing the protein concentration both increase the specific rotation. The value of 94” (Table III) was obtained with a solution of an initial pH of 7.4 and a protein concentration of 6 to 7 gm. per 100 cc. of solution. Actually we could have predicted the observed rotation on the basis of our previous experiments.

TABLE III

Dependence of Specific Optical Rotation, [a], of Alkaline, Heat-Denatured Egg Albumin, Solution D-3, upon State of Ionization. of Protein.

10 Cm. Polarimeter Tubes. X = &61 A.

Final pH

7.83 8.06 8.36 8.70 9.40 9.69

Protein concentration per 100 cc. solution a 14

gm. degress d.3@We8

3.55 -3.31 -93.2 3.57 -3.33 -93.2 3.55 -3.31 -93.2 3.50 -3.30 -94.3 3.51 -3.30 -94.0 3.52 -3.31 -94.0

Although nothing certain can be said concerning the significance of the form of the [OL] to pH or [o(l to concentration relations, nevertheless they are valuable to the end for which they were originally determined; namely, for t,he quantitative characteriza- tion of heat-denatured egg albumin. Redefinition of heat-de- natured egg albumin in terms of solubility or the nitroprusside reaction (Harris, 1923) may now be refined by a statement of the numerical value of the optical rotation under well defined con- ditions, and one may assume that samples of denatured egg albu- min of the same specific optical rotation are probably identical, whereas samples possessing markedly different values for this property are certainly distinct chemical entities. Undoubtedly

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12 Heat-Denatured Egg Albumin Rotation

this same property may be used in the characterization of other denatured proteins.

SUMMARY

1. The rotatory power of alkaline, heat-denatured egg albumin varies in an orderly manner with the time and temperature of heating and approaches a definite limiting value.

2. The limiting value is primarily a function of the hydrogen ion and protein concentrations of the solution during the actual heating.

3. Changes of either hydrogen ion or protein concentration following heating have a slight or negligible effect upon the rotatory power.

4. The optical rotatory power is the only property which at present is known to be suitable for quantitatively characterizing a denatured protein.

5. In general, the pH of an egg albumin solution changes ma.rkedly as a result of heating. Data are given showing the relation of this pH change to the pH of the original solution.

In conclusion the author wishes to express his appreciation to Professor James W. McBain for criticism and advice.

BIBLIOGRAPHY

Chick, H., and Martin, C. J., Kolloidchem. Beihefte, 5,49 (1913). Frisch, J., Pauli, W., and ValM, E., Biochem. Z., 164, 401 (1925). Harris, 5. J., Proc. Roy. Sot. London, Series B, 94, 426 (1923). Holden, H. F., and Freeman, M., Australian J. Exp. Biol. and &fed. SC., 7,

13 (1930). Quagliariello, B., Biochem. Z., 44, 157 (1912). SjGgren, B., and Svedberg, T., J. Am. Chem. Sot., 62, 5187 (1930). &rensen, S. P. L., and H~yrup, M., Compt.-rend. trav. Lab. Carlsberg, 12,

12 (1917). Mreneen, S. P. L., and Jtirgensen, E., Biochem. Z., 31,397 (1911). &reneen, S. P. L., and S#reneen, M. H., Compt.-rend. trav. Lab. Carlsberg,

16, No. 9 (1925).

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H. Albert BarkerHEAT-DENATURED EGG ALBUMIN

THE OPTICAL ROTATORY POWER OF

1933, 103:1-12.J. Biol. Chem. 

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