Denaturation studies on prostatic acid phosphatase

Denaturation Studies on Prostatic Acid Phosphatase] Morris London, Paul Wigler2 and Perry B. Hudson3 From the Departments of Biochemistry and Urology, Francis Delajield Hospital and the Institute of Cancer Research, Columbia University, College of Physicians and Surgeons, New York, New York Received

November

20, 1953

INTRODUCTION

We have found human prostatic acid phosphatase to be a stable enzyme, and in acetate buffers it is stable outside the refrigerator. This is a study of the conditions of stability of the enzyme toward heat and PH. For simple organic reactions a twofold change in rate for a 10°C. change in temperature is a general rule (1). For protein denaturation, a 40-fold change in reaction rate for the same temperature difference is quite common. For this reason thermodynamic measurements, valuable for studying the nature of proteins, are difficult to make, since the large QIOlimits the temperature range over which measurements can be made. For enzymes, the rates of denaturation are dependent on both temperature and pH. METHODS A prostatic acid phosphatase preparation purified 300-fold according to the method of London and Hudson (2) was used in most of the studies. The preparation had a starting concentration of 0.50/, protein, and was used after dialysis against distilled water. It was diluted ten-fold into the buffering system used in each inactivation study, and then exposed to one of the three constant temperatures 50.3, 54.4, and 56.4”C. At accurately measured times, O.l-ml. samples of these solutions were taken, and diluted again into 0.2 M acetate4 buffer at oH 1 This investigation was supported by a grant from the Damon Runyon Memorial Fund. 2 Part of this paper is from the Master’s Thesis of Paul Wigler, Brooklyn College, 1952. 3 Damon Runyon Senior Clinical Research Fellow. 4 A 0.2 M buffer implies the molarity of the anion used, the sum of dissociated and undissociated anion. 236

PROSTATIC

ACID

PHOSPHATASE

237

5.0, containing 0.02% egg albumin as a stabilizing agent. These dilutions were refrigerated at least 24 hr. before being assayed at a final dilution of 2500-fold. Samples heat inactivated until leas than 30% of initial activity remained were diluted only lOOO-fold. The method of assay and unit of activity used were both the same as that used in the purification (2), a modification of the method of Shinowara, Jones, and Reinhart (3). In preliminary work, eight samples under varying buffer conditions and temperatures, without stabilizing agents, were run in 2500-fold dilution in 50-ml. volumetric flasks. Concentrated egg albumin was added to each flask immediately after heating so as to bring its concentration to 0.02% protein. These samples were assayed for activity immediately after heating because several solutions were at pH values where there was a lack of stability at 5°C. All data concerning ion concentration and pH were obtained at 25”C., and, from these, values can be calculated or measured for higher temperatures. The variation of pH with temperature (from 25 to 50°C.) was neglected. RESULTS

The denaturation follows first-order kinetics as indicated by the equation In (ar/ai) = - kAt, where ai and uf are the initial and final activities

TIME

Fro.

IN

HOURS

1. Presentation of typical heat-denaturation curves demonstrating order kinetics with regard to concentration. A, 0.18 M acetate buffer, pH 4.5, 50.3”C. 0, 0.050 M citrate buffer, pH 5.0, 50.3”C. 0, 0.90 M (NHd)&SOd in 0.18 M acetate buffer, pH 5.0, 50.3”C. 6, 0.18 M citrate buffer, pH 5.0, 50.3”C. El, 0.90 M succinate buffer, pH 5.0, 50.3%. n , 0.18 M succinate buffer, pH 5.0, 56.4”C.

first-

238

M.

LONDON,

P. WIGLER

AND

P.

B.

HUDSON

measured for time interval At. To demonstrate that the denaturation follows first-order kinetics closely, in the region of protein concentration used for these studies and in optimally protected systems (egg albumin), several representative denaturation curves are presented in Fig. 1 (also see Fig. 3). The eight samples heat denatured at 2500-fold dilution provided curves almost identical with those denatured under identical conditions at the higher concentration. The selection of the higher concentration for the work in this paper was made because it provided the smoother curves. ‘3”8r65-

43-

2I:;.7.6 .5 2 -4L .3 2 E0 .es 0 8 _ G o;Q :os.07s .063 .05.t .04‘; 0 .OJI 3 no&?I 3.0

I 3.5

I

40

4.5

I1 5.0

5.5

PH

0 rates 0 tate

FIQ. 2. A, B, and C were run at 56.4,52.4, and 50.3”C., respectively. represents constants obtained without albumin protection. Inactivation were so great th&interference by surface effects was negligible. represents constants obtained with albumin protection. Only 0.18 M acebuffers were used.

PROSTATIC

ACID

239

PHOSPHATASE

too _ 90. b .c, 60. .c, '70. ;50lz 50, .C 40 .; =; 30 2 ;20 3 Q

A

IO

I

1

6.67

13.3

Time

I

2 0.0

1

26.6

33.3

in Hours

FIG. 3. Inactivation-rate curves at four different buffer. Symbols used as follows: 0.045 M, 0; 0.09 W,

corweut rations

of acetate

q ; 0.18 21, 0 ; 0.36 M, A.

Figure 2 shows a plot of points for the inactivation-rate constants, at three temperatures, for several pH values using the first-order rate constants found graphically, and expressed in reciprocal hours. The constants are plotted on a logarithmic scale. Since most of the work presented involves the use of acetate buffers, it is of interest to know the role acetate ion plays in influencing the inactivation rat,e. Figure 3 presents inactivation curves for four concentrat’ions of acetic acaid-sodium acetate buffer at pH 5.0. In Table I are presented inactivation-rate constants for succinate, citrate, and ammonium sulfate-acetate buffers compared with the acetate buffer system. Thus, variations in stability were produced with different salts in the buffer system. A more detailed study was made to determine the role of anion concentrations in the denaturation reaction. For comparison, sodium salts were used except for (NHh)zSOd , where the sodium salt presented a solubility problem. Figure 4 shows a plot of first-order inactivation-rate constants against molarity of the anion used. Applying the Arrhenius equation, log k = g+,

240

M.

LONDON,

P. WIGLER

AND

TABLE First-Order

HUDSON

I

Inactivation Rate Constants” for Different Buffer Systems and Temperatures

Buffer

sodium

P. B.

acetate

succinic acidsodium succinate

acetic acidsodium acetate a Constants are obtained b Done without albumin

PH

Calculated ionic strength

kmr.

ksw

4.0 5.0 5.8 4.0 5.0 5.8

0.036 0.13 0.20 0.08 0.20 0.44

0.42 0.047 0.69 2.54 0.191 0.98

1.29 0.15 2.02 5.76 0.40 2.28

5.9” 0.65 8.5b 20.4 2.32 11.1

5.0

0.60

0.29

0.97

4.57

5.0

2.73

0.25

0.62

in reciprocal protection.

kso.ao

2.95

hours.

Molarity

FIG. 4. Stability of enzyme in different buffers with varying concentrations. Sulfate and chloride systems were in 0.18 M acetate buffer. The contribution to inactivation by dilute acetate can obviously be neglected. Symbols used as follows: acetate, l ; sulfate, A; citrate, 8; chloride, n ; succinate, 0.

PROSTATIC

ACID

PHOSPHATASE

241

r x IO’ 5. Curves showing constant energy of activation for denaturation. For convenience, only several representative curves are shown. All systems studied had almost the same slope for given pH and buffer system. A: 0.18 M succinate, pH 4.0 B: 0.18 M succinate, pH 5.8 C: 0.18 X acetate, pH 5.8 D: 0.18 M acetate, pH 4.0 E: 0.18 M citrate, pH 5.0 P: 0.9 M (NH,)zSOa in 0.18 M acet.ate, PI-I 5.0 G: 0.18 M succinate, pH 5.0 H: 0.18 M acetate, pH 5.0 FIG.

where k is t’he reaction rate constant, A.E;the energy of activabion, R the gas constant, and T the absolute temperature, to the inactivat’ion kinetics, enough data are available to plot log k values vs. 1 ,/T and obtain AE/2.3R values. See Fig. 5. DISCUSSION

Enzymes, a,s a class of proteins, lend themselves to denaturation studies. Solubility and chemical change (viz. change in -SH) are the primary methods (4, 5) used for studying alterations in proteins. Enzymatic activity (as in t’he case of the prostatic acid phosphatase) wn be easily and accurat’ely measured, and offers many advantages in

242

M.

LONDON,

P. WIGLER

AND

P. B.

HUDSON

studies of the kinetics of denaturation. Once assurance has been secured that only one molecular species is responsible for the activity studies, and that impurities do not affect the reaction rates, the enzyme can be studied in the impure state. Activity measurements for enzymes can be made with quantities of protein so small that it is at present impossible to measure protein content or changes in free SH groups. The correlation of loss of biological specificity with physical and chemical changes a.ids in establishing the picture of the denaturation. Dilution (over a 250-fold range of variation) is not a factor governing the kinetics of enzyme denaturation, at lea.st for this enzyme system; when surface denaturation is not a factor (2). This fact seems to be contrary to the findings of other investigators for another enzyme who have not attempted to control surface factors (6). The justification for using acetate buffers generally is that acetate seems to alter denaturation constants least of all system tested. See Fig. 4. The optimal stability of the enzyme is found to be close to pH 4.7. From Fig. 2 it can be seen that the behavior pattern of the enzyme in its impure form present,s the general pa-temperature inactivation characteristics observed with crystaJline proteins (7, 8). It should be noted that A log k/A pH is almost constant in all three curves, except for the region of maximum stability. Hypothesis The evidence suggests the following general picture, in which the isoelectric point (9) would fad1 in the region of maximum stability, and that Ic oc [l/H+]“’ for the alkadine region and k a [H+]“” for t’le acid region. C1 and CZ a.re a,pproximately equal in this case (1.5 f .17). The law of mass action may be applied to the system in which [H+] is varied, considering as a simplified model of the states of the protein, the system: p- H+ -H+

po H+ -Hf

p+.

The H+ interacts with the free acidic and basic groups of the protein, which are largely free a.mino and carboxyl groups. P+ and P ma,y be multivalent, and may represent many forms with varying amounts of net charge. PO (or in general some fraction or fractions possessing minimal net charge) ma,y. be considered as the most stable forms. An excess of similar charges can strain the internally organized structure of many forms of the protein molecule by stretching

PROSTATIC

ACID

243

PHOSPHATASE

it. This coulombic stretching of an excessively negatively or positively charged form in addition to the normal stretching of the thermal vibrations that a macromolecule can possess may disrupt internal organization beyond the ability to recover, producing denaturation. AE represents the limiting thermal energy absorbed in addition t.o coulombic stretching before the process becomes irreversible. The AE value, as seen from Fig. 5, is constant. There are at least two forms of the enzyme protein, a positively charged form and a negatively charged form normally stretched to such a point that they are the minimal stable forms existing in solution. The positively charged form predominates in the denaturation process in the acid region. The negatively charged form predominates in the process in t,he alkaline region. Both forms are about equally stretched by coulombic forces. Only these minimally stable, highly charged forms (both positive and negative) are required for the denaturation. At the point of maximum stability these forms exist to only a slight extent and thus the enzyme is stable. The denaturation process occurs only because these unstable forms exist at least in small quantity under all conditions of pH, and are in dynamic equilibrium with all other existing forms. Since the pathway of denaturation is fixed to two equivalently stretched but oppositely charged forms, it explains the constant value of AE. The increasing rate with deviation from the pH of maximum stability occurs because of the numerical increase of one of these forms. The alteration of protein charge of these forms does not have to be accomplished by H+ alone, since AE is fixed for all anion systems tested, The reaction, e.g., coo-

cood

+ citrate’ \

NHa+

G P

/ \

.(NHI

citrate)--

may take place, producing the negatively charged form. This mechanism differs from that proposed by Levy and Benaglia (8). They contend that the critical factor in the disruption of the protein molecule (denaturation) is “ . . . proton-sensitive hydrogen bonds involved in specific biologically imposed chelations. Loosening of a limited number of these does not necessarily lead to denaturation but does decrease the amount of thermal energy which- must be absorbed to break away from the metastable native configuration. Once past the limit,, t,he specific

244

M.

LONDON,

P. WIGLER

AND

P. B.

HUDSON

structure of the protein is lost and the peptide chain unfolds or rearranges to the denatured state.” This hypothesis cannot account for the increased inactivation rates with constant thermal energy absorption for denaturation for dilute buffer systems such as succinate or citrate. Ionic strength, as seenfrom Table I, does not correlate with denaturation, and it is not significant in the reaction for dilute buffer systems. To have maintained ionic strength would have required sacrificing anion or pH control. For concentrated salts as in the case of sulfate(-‘) and citra)e’-3’ where ionic strength increased rapidly with concentration, there is an inversion of the increasing denaturation with concentration (see Fig. 4). The stability acquired with high salt concentrations may be explained by the dehydration (salting out) of the protein which is a property of multivalent ions. Proteins become more heat-stable when dehydrated, and dry crystalline enzymes are often stable at 100°C. If the preceding hypotheses properly explain protein denaturation, then it follows that, since the value d log k/d pH is a constant (C, or C,) on either side of the region of maximum stability, this new constant ought to be a measure of the dissociation of acidic or basic groups, since their presence regulates the H+ and anions that can be taken on or given off (hence the rate of build-up of coulombic forces with pH change). The constant should characterize properties such as solubility and surface activity, which are strongly affected by charge. The fact that Cl and CZare about equal supports the idea that certain molecular forms, highly stretched by coulombic repulsion of like charges, are the ones which undergo denaturation, since the critical extent of coulombic stretching is independent of whether the net charge is positive or negative. The values for AF* and AS* have been calculated from equations derived from absolute reaction rate theory (10). The AH*6* 6 is high like that of most proteins, and is higher than the corresponding values ob6AE and AH* can be considered identical. The relationship is A,!3- RT. The relatively high APE and low T make RT an almost negligible

AH* = term. 6 The values for AE have been calculated from the slopes shown in Fig. 5. They range from 84.9 to 93.1 kcal./mole and average 88.7 kcal./mole. The AE derived from curve A was omitted. The value is 75.4 kcal./mole; however, eliminating the point on that curve corresponding to 56.4%. would produce a new slope corresponding to AE of 84.1 kcal./mole. The justification for ignoring that denaturation constant is that the last measurable residual enzyme activity during denaturation occurred after 5 min. of heating.

PROSTATIC

ACID

TABLE II Comparison of Thermodynamic ~ AIf* kcal./mole

System

245

PHOSPHATASE

Values / AS* al./ mole/deg.

1 AF’so.rx kcal/mole

/

Reference

Egg albumin, in dil. HCI (from k”, derived constant) Egg albumin (k’ derived constant) Ricin, in mixed varying buffers (from K*, , derived constant)

36.7 89.3

48.5 168.5

21.0 34.8

Nord (4) Do. Levy and Benaglia

Urease (dry heat) Hemoglobin (water, pH 6.8) Trypsinkinase (in 24% glycerol, pH 6.5) Prostatic acid phosphatase, pH 5.0, 0.18 IV acetate, 50.3-56.4”C. Same enzyme, pH, and temp., 0.9 M (NHI)zS04 , 0.18 M acetate Same enzyme, pH, and temp., 0.18 M citrate Same enzyme, pH, and temp., 1.52 M succinate

20.8 76.3 44.3

-22 152 57.6

27.9 27.2 25.7

Setlow (11) Lewis (12) Pace (13)

(8)

go.o ~225

17.3

This paper

86.4

/ 223

14.6

1 This paper

92.5

241

14.6

~ This paper

-

13.2

This paper

tained for most enzymes studied. Table II shows values calculated for the denaturation of prostatic acid phosphata,se studied under different csonditions, and makes comparisons with other enzymes and proteins. The AF* values reported in the literature were convert~ed to values (‘orresponding t’o 50.3%. ACKNOWLEDGMENTS

To Drs. John M. Reiner and Irwin B. Wilson u-e are indebted for many valuable suggestions and theoretical discussions. Miss Rosemary McHugh we thank for supplying some of the data presented here. SUMMARY

The heat denaturation of prostatic acid phosphatase has been studied in a controlled system, as a function of a number of primary environmental variables. The factors of ionic strength and variations in protein concentration were shown to be negligible or controllable. The nature of anion in the buffer system profoundly affects the denaturation rate. The multivalent anions accelerate the rate rapidly for

246

M. LONDON,

P. WIGLER

AND P. B. HUDSON

increasing low molarities, whereas the rates decrease after passing through a maximum for higher concentrations. Ions of lower valency give rates increa’sing with concentration, with no maxima. The activation energy is constant regardless of buffer or pH used. REFERENCES 1. GETMAN, F. H., AND DANIELS, F., “Outlines of Physical Chemistry.” John Wiley and Sons, New York, 1943. 2. LONDON, M., AND HUDSON, P. B., Arch. Biochem. and Biophys. 46,141 (1953). 3. SHINOWARA, G. Y., JONES, L. M., AND REINHART, H. L., J. Biol. Chem. 143, 921 (1942). 4. GIBBS, R. J., BIER, M., AND NORD, F. F., Arch. Biochem. and Biophys. 36, 216 (1952). 5. MIRSKY, A. E., AND ANSON, M. L., J. Gen. Physiol. 18,307 (1935). 6. CASEY, E. J., AND LAIDLER, K. J., J. Am. Chem. Sot. 73, 1455 (1951). 7. CUBIN, H. K., Biochem. J. 23,26 (1929). 8. LEVY, M., AND BENAGLIA, A. E., J. Biol. Chem. 186,829 (1950). 9. DEROW, M. A., AND DAVISON, M. M., Science 118, 247 (1953). 10. GLASSTONE, S., LAIDLER, K. J., AND EYRING, H., “The Theory of Rate Processes.” McGraw-Hill, New York, 1941. 11. SETLOW, R. B., Arch. Biochem. and Biophys. 36,328 (1952). 12. LEWIS, P. S., Biochem. J. 20, 965 (1926). 13. PACE, J., Biochem. J. 26, 422 (1931).