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Relative reactivities of chemically modified turkey ovomucoids
ARCHIVES
OF
BIOCHEiWSTRY
Relative
AND
Reactivities
BIOPHYSICS
of Food
64-71
of Chemically
IMAHAVIR Department
113,
Science
Modified
M. SIMLOT
Turkey
ROBERT
AND
and Technology, Received
(1966)
University June
Ovomucoids’
E. FEENEY
of California,
Davis,
California
15, 1965
The interactions between avian ovomucoids and bovine trypsin or bovine a-chymotrypsin were found to occur at different rates. The rates are dependent upon the types of ovomucoid and enzyme. In the case of turkey ovomucoid, which is an inhibitor of both trypsin and a-chymotrypsin, the reaction with trypsin was approximately three times more rapid than the reaction with a-chymotrypsin. Chemical modification of either the turkey ovomucoid or the enzymes could change the rate of interaction. Chemical modifications of the ovomucoid by several reagents which formed more acidic derivatives increased by several fold the rate of interaction with a-chymotrypsin. These modifications were acetylation, carbamylation, succinylation, and iodination. Amidination of the ovomucoid, which caused no significant change in charge, did not change the rate.
Avian egg white ovomucoids have the biochemical property of inhibiting the proteolytic enzymes bovine trypsin and bovine cr-chymotrypsin. The ovomucoids from different avian species show interesting differences in specificities. Chicken ovomucoid inhibits trypsin; golden pheasant ovomucoid inhibits cr-chymotrypsin (with a very weak affinity for trypsin) ; and turkey ovomucoid inhibits both trypsin and ar-chymotrypsin, and with the inhibitory activities apparently being mutually independent from one another (l-3). Stevens and Feeney (3) recently reported differences in the effects of chemical modifications. They demonstrated that the inhibitory activity of turkey ovomucoid for trypsin was destroyed by acetylation or carbamylation, while the inhibitory activity against cu-chymotrypsin was unaffected. In addition, the inhibit’ory activities of chicken ovomucoid for trypsin and golden pheasant ovomucoid for ol-chymotrypsin were not 1 This work was supported in AI-03484 and HD-0122 from the tutes of Health. Presented in part Annual Meeting of the American logical Chemists, Chicago, Illinois, 1964.
part by grants National Instibefore the 48th Society of BioApril 12-17, 64
destroyed by such treatments. Although differences in amino acid composition of these three ovomucoids were found, no major differences in physical or chemical properties were observed (3). In the present studies, the chemical modifications of turkey ovomucoid were extended to other reagents reacting with amino groups. Particular at’tention has been given t’o the effects of such changes on the rates of interaction of turkey ovomucoid with bovine trypsin or cr-chymotrypsin. MATERIALS Salt-free crystalline preparations of bovine trypsin (2X crystallized) and cu-chymotrypsin (3X crystallized) were purchased from the Nutritional Biochemical Corporation and Worthington Biochemical Company, respectively. Activities of these preparations, as determined by inhibitory assays with chicken and turkey ovomucoid (1, 3), were approximately 75y0 of enzymically active trypsin and 95y0 of enzymically active wchymotrypsin. Spectrophotometric determination of the operational normality of the chymotrypsin with N-transcinnamoylimidazole according to the procedure of Schonbaum et al. (4)) also gave a value of 95%. Weights and molar ratios expressed were corrected for these partial activities (1). Approximate values used for molecular
CHEMICALLY
MODIFIED
weights were 28,000, 23,000, and 23,000 gm for ovomucoid, trypsin, and chymotrypsin, respectively. Ovomucoids were prepared by trichloroacetic acid-acetone precipitation (5) followed by chromatography on CM-cellulose2 and DEAEcellulose (1, 3). The trypsin substrate, TAME, was purchased from the Mann Research Laboratories. The chymotrypsin substrate, BTEE, was synthesized by benzoylation of n-tyrosine ethyl ester hydrochloride (3). Enzyme assays. Enzymic assays were done spectrophotometrically according to the methods described by Rhodes et al. (2, 6) and Feeney et al. (1). The indicator was 0.27, m-nitrophenol. The enzymic activity was measured by the rate of change in percentage transmission; a Beckman DB recording spectrophotometer was used. Rate measurements. The rates of inhibition were determined from the residual enzymic activity (6). Routinely, 0.3 ml of a solution containing 20-24 rg of enzyme in 0.004 M acetic acid and 0.02 M CaC12 was added to 0.7 ml of a solution containing 1015 pg of inhibitor in 0.006 M tris buffer, pH 8.95. The final pH of the mixture was 8.15-8.25. The mixture was incubated for various periods of time, and then 2 ml of a solution containing substrate (0.01 M), indicator (0.2%), and buffer (0.006 M tris, pH 8.2) was added. Changes in absorbancy at 395 mp were followed by the recorder. Confirmatory determinations of the number of moles of substrate hydrolyzed were made by direct measurement of the amounts of acid formed at constant pH; a Di-functional Recording Titrator from the International Instrument Co., Canyon, California, was used. The pH was maintained constant by the automatically controlled addition of dilute alkali. The techniques for determinations of rates by delay times were essentially the same as described by Green (7) for various trypsin inhibitors. In one type of determination, 0.4 or 0.5 ml of pH 8.2 buffer and 2 ml of the substrate-indicator-buffer solution were added to 0.3 ml of enzyme solution (20-24 pg). This was followed immediately by the addition of 0.2 or 0.3 ml of the ovomucoid solution (10-35 fig of ovomucoid). Changes in the percentage transmission were recorded. In a second type of determination, the order of addition of the ovomucoid and enzyme was reversed: An appropriate amount of the enzyme solution was added * Abbreviat,ions used: CM-cellulose, carboxymethyl cellulose; DEAE-cellulose, diethylaminoethyl cellulose; TAME, p-tosyl arginine methyl ester; BTEE, benzoyl-tyrosine ethyl ester; TCAacetone, trichloroacetic acid-acetone (1 volume of aqueous 0.1 Jf trichloroacetic acid + 2 volumes of acetone).
TURKEY
OVOMUCOIDS
65
to a mixture of substrate-indicator-buffer solution and the inhibitor and the changes in the percentage t,ransmission were recorded. Acetylation.3 Proteins w-ere acetylated essentially according to the method of Fraenkel-Conrat (8). Two hundred mg of enzyme or ovomucoid was dissolved in 2 ml of half-saturated solution of sodium acetate and cooled in an ice bat’h. To the cooled solution, 0.02 ml of acetic anhydride was added in five equal increments over a period of 1 hour. The mixture was dialyzed, centrifuged to remove insoluble material? and lyophilized. When enzymes, especially trypsin, were acetylated, the yield of products was only 507,. Purification by chromatography was as recently described (3). The degree of acetylatioii was determined by measuring the change in the number of free amino groups upon acetylation. The number of free amino groups was determined by the ninhydrin method (8) as standardized against the Van Slyke method with selected samples (3). SuccinyZation.4 This was done according to the method described by Habeeb et al. (9) and Buttkus et al. (10). A 20-fold excess of solid succinic anhydride was added to a 1.5 or 10% solution of ovomucoid in 57, sodium bicarbonate. The reagent was added at room temperature at intervals over a ZO-minute period, with const,ant stirring. The mixture was left standing another 20 minutes, after which it was dialyzed against water and then lyophilized. In these studies, the two concentrations of ovomucoid, 1.5 and lo%, were used to obtain low and high succinylated derivatives. The degree of succinylation was determined as for acetylation. Reaction with ethyl acetimidate hydrochloride.6 Amidination was done according to the procedure of Wofsy and Singer (11) by reacting the protein with ethyl acetimidate hydrochloride. The reagent 3 The degree of acetylation of the amino groups of the particular preparations used in these experiments were: trypsin, 7370; chymotrypsin, 66%; turkey ovomucoid, 707c. 4 As determined by the ninhydrin method (8), the amount of succinylation of amino groups was 157c for the low succinylated and 60% for the high succinylated preparations, respectively. j The amount of modification of amino groups by amidinat,ion was calculated by two methods. The ninhydrin method (8) gave a value of 48% for the decrease in amino groups; the automatic amino acid analyzer gave a value of 60% for the decrease in lysine. In the amino acid analysis, a new peak of a modified amino acid was located close to histidine, between histidine and arginine. The amount of material in the new peak, approximately account,ed for the loss in lysine.
SIMLOT
66
AND FEENEY
was prepared as described by McElvain and Nelson (12). Five ml of a lyO solution of ovomucoid in 0.1 111 borate buffer, pH 8.5, were mixed with 0.25 ml of a 0.1 111 solution of ethyl acetimidate hydrochloride in 2 III NaOH, and the pH was adjusted to 8.4. After the mixture was cooled, the pH was maintained in a pH stat for 2 hours. Then it was dialyzed and lyophilized. Zodination. Iodination was done as described by Stevens and Feeney (3) for a high level of iodination. A 10% solution of turkey ovomucoid at pH 8.5 was treated with iodine solution in potassium iodide. A sample of the iodinated ovomucoid was also acetylated with acetic anhydride as described earlier. RESULTS
Efects of chemical naod$cation of b-key ovomucoid. Table I summarizes the inhibitory activities of several differently modified ovomucoids. All three reagents which modify amino groups inactivated t,he trypsin inhibitory activity without affecting the chymotrypsin inhibitory activity. These reagents essentially caused no change in charge (amidination), a single change in
TABLE ACTIVITIES
I
OF CHEMICALLY TURKEY OVOMUCOID
Reagents
1. Acetic anhydridea 2. Succinic anhydrideb Low High 3. Ethyl acetimidate 4. Iodine in HI soln.a 5. Iodine + acetic anhydride”
MODIFIED
“rp Inhibitory actwity of contra: Trypsin
Chymotrypsin
<5
100
18 <5 <5
100
100 100 100
100 100
a In agreement with Stevens and Feeney (3). b Low succinylation was obtained with lCyO protein solutions and high succinylation with lO7C protein solutions. Experimental procedure is given in the text. c Ovomucoid was first iodinated with an iodine solution in KI in alkaline solution, dialyzed and lyophilized. The lyophilized iodinated ovomucoid was then acetylated with acetic anhydride.
gradual. The initial slopes of the plots depended also upon the ratio of enzyme to negative charge per amino group substituted ovomucoid. A low ratio gave sharper slopes (acetylation) or two changes in negative in the initial period, and vice versa. The rate charge per amino group substituted (suc- of interaction between chymotrypsin and cinylation). Iodination, which strongly inturkey ovomucoid was not affected, howcreasesthe charge, did not cause any losses ever, by the presence of four times the of activity while iodination combined with amount of trypsin as chymotrypsin. acetylat,ion inactivated only the inhibitory The rates of reaction of duck ovomucoid activity for trypsin. These results extend with trypsin and chymotrypsin were similar the previous findings of Stevens and Feeney to the rates of turkey ovomucoid, but (3) w&h carbamylat’ion, acetylation, or ovomucoid from the ring-necked pheasant iodination. They indicate that 6he change react,ed rapidly with both chymotrypsin in charge introduced by the modification is and trypsin. Chicken ovomucoid reacted not t#he cause of inactivation of the inhibirapidly with trypsin, and golden pheasant tory activit,y against trypsin. ovomucoid reacted rapidly with chymoRelative rates of yeaction with trypsin and trypsin. chymotrypsin. When the length of incubaConfirmatory estimations of the rates of tion of the mixtures of inhibit’or and enzyme hydrolysis of substrate by direct det’erminaprior to addition of substrate was varied, tion of the amounts of alkali consumed in trypsin and chymotrypsin reacted with avian the reaction were comparable, in general, to ovomucoids at different rates (Fig. 1). With the rates obtained by the spectrophottoincreased time of incubation, the residual metric method.6 enzymic activity decreased until maximal 6 The reaction as shown in Fig. 1 appears to be inhibition was reached. In general, this of second order, but the calculation of rate conoccurred considerably later with chymo- stants is difficult with this type of data. The orders trypsin than with trypsin. The activity of of the reactions and the calculations of rate contrypsin decreased rapidly, while the decrease stants are presently under investigation by more in activity of chymot’rypsin was more direct methods.
CHEMICALLY
MODIFIED
TURKEY
Tryp-Ovomumd
(18.10)
vowcold
60
120
TIME OF INCUBATION
180
240
OF ENZYME (seconds’
67
OVOMUCOTDS
(22:15)
300
360
420
AND INHIBITORS
FIG. 1. Times required for inhibition of trypsin and a-chymotrypsin by turkey ovomucoid. Numbers in parentheses refer to weight ratios of trypsin (Tryp) or chymotrypsin (Chymo) to turkey ovomucoid. Enzyme and ovomucoid were incubated together in pH 8.2 buffer for the periods indicat.ed, and then mixed with a solution containing substrate, indicator, and buffer. Changes in percent transmission at 395 mp were followed immediately on the recording spectrophotometer. Details are given in text.
Effects of acetylation of turkey ovomucoid on enzymes. In our earlier studies we showed that acetylation of turkey ovomucoid destroyed its capacity to inhibit trypsin but did not, destroy it,s capacity to inhibit a-chymotrypsin. This inactivation of the inhibitory activit,y for trypsin has been confirmed in the current studies. However, it, also has been found bhat the inhibitory activity for chymotrypsin was affected. The effect was expressed primarily as a 2-3fold increase in rate of inhibition (Fig. 2). This gave the illusion of a much greater comparative inhibitory activity of the acetylated ovomucoid for chymotrypsin if the determinations were performed without allowing sufficient time for the relatively slow reaction bet#ween the unmodified inhibitor and the enzyme to occur. A more
rapid reaction of the acetylated ovomucoid also was apparent in “delay t.ime” determinations (Fig. 3). A slightly increased total apparent inhibitory activity of the acetylated ovomucoid for chymotrypsin also was observed consistently. This apparent activity was approximately 5% greater than that of the unmodified ovomucoid on a stoichiometric basis, and was considered a reflection of higher affinity for the enzyme. Acetylated trypsin was essentially uninhibited by turkey ovomucoid, confirming similar findings with chicken ovomucoid (13). Acetylated chymot’rypsin was still inhibited by turkey ovomucoid, however, but the reaction was slow (Figs. 2 and 3). The apparent relative rates of interaction for acetylated turkey ovomucoid and acetylated chymotrypsin were in the following order (beginning with the most rapid):
GS
SIMLOT
I 60
I 120
TIME OF INCUBATION
AND
FEENEY
I 180
240
OF ENZYME (seconds)
300
I 420
360
AND INHIBITOR
FIG. 2. Effects of acetylation on times required for inhibition of ar-chymotrypsin by turkey ovomucoid. Weight ratios of chymotrypsin to turkey ovomucoid were 22:15. Abbreviations used: Chymo, chymotrypsin; AcChymo, acetylated chymotrypsin; TO, turkey ovomucoid; AcTO, acetylated turkey ovomucoid. Enzyme and ovomucoids were incubated together in pH 8.2 buffer for the periods indicated and then mixed with a solution of the substrate, indicators and buffers. The changes in percent transmission at 395 mp were then immediately followed on the recording spectrophotometer. Details are given in text.
acetylovomucoid-chymotrypsin; ovomucoidchymotrypsin; acetylovomucoid-acetyl-chymotrypsin; and ovomucoid-acetylchymotrypsin.6 Delay time assays. Green (7) described this technique for measuring displacement of the substrate, by the inhibitor, from the enzyme-substrate complex. In his experiments, the inhibitor was added to mixtures of the enzyme and substrate and the times required for inhibition to occur were noted. It is evident from Figs. 3 and 4 that there was a demonstrable time required for the occurrence of inhibition, confirming Green’s result,s with chicken ovomucoid. When the order of addition of the enzyme and ovomucoid was changed (Figs. 3 and 4), however, no significant’ differences were observed
in the times required for inhibition to occur. There thus was no evidence that the inhibitor displaced the substrate from the enzyme-substrate complex. The delay time assays also were used to study the effects of modification on the relative rates of interaction with the enzymes (Figs. 3 and 4). All three modifications of ovomucoid directed at the amino groups inactivated the antitryptic activity, as also was found by the direct methods (Fig. 1; Table I). However, major increases were found in rates of inhibitions of iodinated derivatives with both trypsin and and amidination did not chymotrypsin, cause an increased rate with chymotrypsin as did acetylation or succinylation. All three modifications
causing
an
increased
negative
CHEMICALLY
MODIFIED
TURKEY
charge, t’herefore, increased the rate of interactions, while the modification causing no significant change in charge (amidination) did not increase hhe rat,e.
OVOMUCOIDS
69
CHYMOTRYPSIN
CHYMOTRY PSIN
AC Chy -TO AC Chy - Ac T O
Chy -TO
so-
1 I I I Po,nt of Add,t,on of Ovomucoid I
Chy -AC T O
I 120
I 240 SECONDS
I 350
I 480
I 360
I 480
80 -
120 SECONDS
55
240
360
TRY PSIN t
AC Try -AC T O AcTry-TO
Try -TO
Potnf of Addltoan I 120
of Ovamuco,d I 240
I
SECONDS
120 SECONDS
240
360
FIG. 3. Times required for inhibition of tryspin and a-chymotrypsin by turkey ovomucoid with “delay time” technique. Abbreviations used: Chy, chymotrypsin; AcChy, acetylated chymotrypsin; TO, turkey ovomucoid; AcTO, acetylated t,urkey ovomucoid; Try, trypsin; AcTry, acetylated trypsin. Weight ratios were 22:15 for chymotrypsin and ovomucoid and 18:15 for trypsin and ovomucoid. Ovomucoid, buffer, indicator and substrate were mixed together and then the enzyme added. The acetylated chymotrypsin had a specific activit.y nearly identical to the original chymotrypsin. acetylated trypsin had a slightly loaer activity than the original trypsin and the plot for acetylated trypsin was similar to the plot given for AcTry-AcTO. Details are given in text.
FIG. 4. Effect of chemical modification on delay time assays. Abbreviations used: Chy, chymotrypsin; TO, turkey ovomucoid; AcTO, acetylated turkey ovomucoid; Try, trypsin; AmTO, amidinated turkey ovomucoid; SucTO, succinylated turkey ovomucoid; ITO, iodinated turkey ovomucoid; IAcTO, iodinated and acetylated turkey ovomucoid. To a mixture of enzyme-buffer and substrate, was added turkey ovomucoid solution within 18-25 seconds and the inhibition recorded on a chart at 395 rnp. The weight ratios were: (a) for chymotrypsin and turkey ovomucoids 22:15; (b) for trypsin and turkey ovomucoids 18:15.
DISCUSSION
Several previous studies have concerned both the mcehanism of inhibition by inhibitors of trypsin and the kinetics of the inhibitory process. Avian ovomucoids have been shown to form physically demonstrable
70
SIMLOT
AND
complexes (1, 2, 14). It has been usually accepted that the mechanism of inhibition involves the formation of a relatively &able complex (15). Early studies with chicken ovomucoid and trypsin, with casein as a substrate, indicated that the inhibition was of t,he “noncompetitive” type (13), but later studies with synthetic substrate were interpreted as showing that a variety of natural inhibitors of trypsin were competitive inhibitors (7, 16, 17). Comparative studies also showed that the pancreatic trypsin inhibitors reacted with trypsin much more slowly than did ot’her inhibitors, including chicken ovomucoid (7). It has been suggest.ed that electrostatic forces are important in the formation of complexes between trypsin and its natural inhibitors (15). In most instances rather large differences were found between the isoelectric point of bhe inhibitor and that of the enzyme. In only one instance mere the isoelectric points close together. This occurred with trypsin and the pancreatic t#rypsin inhibitor, and the reaction between them was relat,ively slow (17). In more recent studies the interaction of trypsin with trypsin inhibitors was found t’o be accompanied by a release of a titrat>able proton (18). Finkenstadt and Laskowski, Jr. (19) have presented evidence for the formation of a covalent intermediate and possible enzymatic hydrolysis of a peptide bond. In the present studies we have found still further differences between t.he properties of avian ovomucoids t’han we have reported previously. In addition to the inactivation of the trypsin-inhibitory activity of turkey ovomucoid by acetylation or carbamylation (without affecting the chymotrypsin inhibitory activity) (3), both amidination and succinylation also were found to produce similar effects. Change in charge due to the modification, therefore, does not appear to be the cause of inactivation. Two other major differences were observed in the dual inhibitory properties of turkey ovomucoid against trypsin and chymotrypsin. One was a large difference between the rates of inhibition of the two enzymes by the ovomucoids. This was of such magnit.ude that, in actual laboratory determination of inhibitory activity by incubating
FEENEY
different mixtures of the enzymes and the inhibitor, an incubation time of only 1 minute usually was satisfactory with trypsin, while at least 3-4 minutes were required with chymotrypsin. The second difference was the effect’ of chemical modification of the ovomucoid on the rat,es of inhibit’ion of the enzymes. Acet’ylation or succinylation increased t.he rat,e of inhibition of chymotrypsin, while amidination did not increase the rate. In addition, iodination, which has no significant effect on the stoichiometry of inhibition of either trypsin or chymotrypsin by turkey ovomucoid (3), also increased the rates of reaction wit,h both trypsin and chymotrypsin. Our studies on relative rates indicate that factors related to charge are important in the interaction of ovomucoids with trypsin or chymotrypsin. However, observat8ions of the rat.es under different, conditions and the inactivation of t.he t,rypsin inhibitory activity by reagents for amino groups which both do and do not affect charge, prove conclusively that ot’her factors are also important. The interactions are thus cornplex and involve a variety of factors, including possibly such general ones like electrostatic and conformational factors and more specific ones like interactions with specific amino acid side chains. The inactivations of the t’rypsin inhibitory activity wit’h reagents for amino groups mdicate a specific involvement of one or more of the E-amino groups of lysine in t’he interaction with trypsin. Such a specific involvement has been suggested by Finkellst’adt and Laskowski (19) for t,he interactions of chicken ovomucoid with Drypsin. These authors have suggested that a covalent linkage is formed with the possible specific hydrolysis at an arginine bond (20). The complexities of the reactions as well as the effects of chemical modifications are so extensive, however, that many st,udies are necessary. For example, it is highly probable that some particular chemical modificat,ion might have opposing effects which might therefore either enhance or decrease the activity depending upon the relative importance of these effects. Since the order of addition of the enzyme and inhibitor in the delay-time studies was
CHEMICAL
MODIFIED
not significant and the inhibitor does not influence deacylation reaction,’ it appears that the procedure measures the rate of association of free enzyme and inhibitor and not the displacement of the substrate by the inhibitor, as suggestedby Green (7). A conventional scheme for the enzymic sequence might be:
in which E, S, ES, and ES* are the enzyme, substrate, initial enzyme-substrate complex, and enzyme-modified substrate complex, respectively; and PI and Pz are the products of hydrolysis of the substrate. According to our interpretation, the inhibitor would form a complex with the free enzyme and the rate of such formation would be much slower than any of the reaction rates between the enzyme and substrate. The formation of a complex between the inhibitor and enzyme, therefore, should influence the course of the reaction between the enzyme and substrate only by relatively slowly removing enzyme from the system. .XKNOWLEDGMENT Appreciation C. Stevens for servations on ovomucoid. 7 In of the ported rate of
is due David T. Osuga and Frits advice and for certain of the obthe properties of acetyl turkey
a personal University that turkey deacylation
communication, Dr. John Mehl of Southern California has reovomucoid does not affect the of cinnamoylchymotrypsin.
TURKEY
OVOMUCOIDS
71
REFERENCES 1. FEENEY, R. E., STEVENS, F. C., AND OSUGA, D. T., J. Biol. Chem. 238, 1415 (1963). 2. RHODES, M. B., BENNETT, N., AND FEENEY, R. E., J. Biol. Chem. 236, 1686 (1960). 3. STEVENS, F. C., AND FEENEY, R. E., Biochemistry 2, 1346 (1963). 4. SCHONBAUM, G. R., ZERNER, B., AND BENDER, M. L., J. Biol. Chem. 236,293O (1961). 5. LINEWEAVER, H., AND MURRAY, C. W., J. Biol. Chem. 171, 565 (1947). 6. RHODES, M. B., HILL, R. M., AND FEENET, R. E., Bnal. Chem. 29, 376 (1957). 7. GREEN, N. M., J. Biol. Chem. 206, 535 (1953). 8. FRAENKEL-CONRAT, H., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. IV, p. 247. Academic Press, New York (1957). 9. HABEEB, A. F. S. A., CASSIDY, H. G., AND SINGER, S. J., Biochem. Biophys. Acta 29, 587 (1958). 10. BUTTKUS, H., CLARK, J. R., AND FEENEY, R. E., Biochemistry 4, 998 (1965). 11. WOFSY, L., AND SINGER, S. J., Biochemistry 2, 104 (1963). 12. MCELVAIN, S. M., AND NELSON, J. W., J. Am. Chem. Sot. 64, 1825 (1942). 13. FRAENKEL-CONRAT, H., BEAN, R. S., AND LINEWEAVER, H., J. Biol. Chem. 177, 385 (1949). 14. RAM, J. S., TERMINIELLO, L., BIER, M., AND NORD, F. F., Arch. Biochem. Biophys. 62, 451 (1954). 15. LASKOWSKI, M., AND LASKOWSKI, Jn., M., Advan. Protein Chem. 9, 203 (1954). 16. GREEN, N. M., AND WORK, E., Biochem. J. 54, 347 (1953). 17. GREEN, N. M., Biochem. J. 66,407 (1957). 18. LEBOWITZ, J., AND LASKOWSKI, JR., M., Biochemistry 1, 1044 (1962). 19. FINKENSTADT, W. R., AND LASKOWSKI, JR., M., J. Biol. Chem. 240, PC962 (1965). 20. FINKENSTADT, W. R., OZAWA,K., AND Las~owSKI, JR., M., Federation Proc. 24,593 (1965).
OF
BIOCHEiWSTRY
Relative
AND
Reactivities
BIOPHYSICS
of Food
64-71
of Chemically
IMAHAVIR Department
113,
Science
Modified
M. SIMLOT
Turkey
ROBERT
AND
and Technology, Received
(1966)
University June
Ovomucoids’
E. FEENEY
of California,
Davis,
California
15, 1965
The interactions between avian ovomucoids and bovine trypsin or bovine a-chymotrypsin were found to occur at different rates. The rates are dependent upon the types of ovomucoid and enzyme. In the case of turkey ovomucoid, which is an inhibitor of both trypsin and a-chymotrypsin, the reaction with trypsin was approximately three times more rapid than the reaction with a-chymotrypsin. Chemical modification of either the turkey ovomucoid or the enzymes could change the rate of interaction. Chemical modifications of the ovomucoid by several reagents which formed more acidic derivatives increased by several fold the rate of interaction with a-chymotrypsin. These modifications were acetylation, carbamylation, succinylation, and iodination. Amidination of the ovomucoid, which caused no significant change in charge, did not change the rate.
Avian egg white ovomucoids have the biochemical property of inhibiting the proteolytic enzymes bovine trypsin and bovine cr-chymotrypsin. The ovomucoids from different avian species show interesting differences in specificities. Chicken ovomucoid inhibits trypsin; golden pheasant ovomucoid inhibits cr-chymotrypsin (with a very weak affinity for trypsin) ; and turkey ovomucoid inhibits both trypsin and ar-chymotrypsin, and with the inhibitory activities apparently being mutually independent from one another (l-3). Stevens and Feeney (3) recently reported differences in the effects of chemical modifications. They demonstrated that the inhibitory activity of turkey ovomucoid for trypsin was destroyed by acetylation or carbamylation, while the inhibitory activity against cu-chymotrypsin was unaffected. In addition, the inhibit’ory activities of chicken ovomucoid for trypsin and golden pheasant ovomucoid for ol-chymotrypsin were not 1 This work was supported in AI-03484 and HD-0122 from the tutes of Health. Presented in part Annual Meeting of the American logical Chemists, Chicago, Illinois, 1964.
part by grants National Instibefore the 48th Society of BioApril 12-17, 64
destroyed by such treatments. Although differences in amino acid composition of these three ovomucoids were found, no major differences in physical or chemical properties were observed (3). In the present studies, the chemical modifications of turkey ovomucoid were extended to other reagents reacting with amino groups. Particular at’tention has been given t’o the effects of such changes on the rates of interaction of turkey ovomucoid with bovine trypsin or cr-chymotrypsin. MATERIALS Salt-free crystalline preparations of bovine trypsin (2X crystallized) and cu-chymotrypsin (3X crystallized) were purchased from the Nutritional Biochemical Corporation and Worthington Biochemical Company, respectively. Activities of these preparations, as determined by inhibitory assays with chicken and turkey ovomucoid (1, 3), were approximately 75y0 of enzymically active trypsin and 95y0 of enzymically active wchymotrypsin. Spectrophotometric determination of the operational normality of the chymotrypsin with N-transcinnamoylimidazole according to the procedure of Schonbaum et al. (4)) also gave a value of 95%. Weights and molar ratios expressed were corrected for these partial activities (1). Approximate values used for molecular
CHEMICALLY
MODIFIED
weights were 28,000, 23,000, and 23,000 gm for ovomucoid, trypsin, and chymotrypsin, respectively. Ovomucoids were prepared by trichloroacetic acid-acetone precipitation (5) followed by chromatography on CM-cellulose2 and DEAEcellulose (1, 3). The trypsin substrate, TAME, was purchased from the Mann Research Laboratories. The chymotrypsin substrate, BTEE, was synthesized by benzoylation of n-tyrosine ethyl ester hydrochloride (3). Enzyme assays. Enzymic assays were done spectrophotometrically according to the methods described by Rhodes et al. (2, 6) and Feeney et al. (1). The indicator was 0.27, m-nitrophenol. The enzymic activity was measured by the rate of change in percentage transmission; a Beckman DB recording spectrophotometer was used. Rate measurements. The rates of inhibition were determined from the residual enzymic activity (6). Routinely, 0.3 ml of a solution containing 20-24 rg of enzyme in 0.004 M acetic acid and 0.02 M CaC12 was added to 0.7 ml of a solution containing 1015 pg of inhibitor in 0.006 M tris buffer, pH 8.95. The final pH of the mixture was 8.15-8.25. The mixture was incubated for various periods of time, and then 2 ml of a solution containing substrate (0.01 M), indicator (0.2%), and buffer (0.006 M tris, pH 8.2) was added. Changes in absorbancy at 395 mp were followed by the recorder. Confirmatory determinations of the number of moles of substrate hydrolyzed were made by direct measurement of the amounts of acid formed at constant pH; a Di-functional Recording Titrator from the International Instrument Co., Canyon, California, was used. The pH was maintained constant by the automatically controlled addition of dilute alkali. The techniques for determinations of rates by delay times were essentially the same as described by Green (7) for various trypsin inhibitors. In one type of determination, 0.4 or 0.5 ml of pH 8.2 buffer and 2 ml of the substrate-indicator-buffer solution were added to 0.3 ml of enzyme solution (20-24 pg). This was followed immediately by the addition of 0.2 or 0.3 ml of the ovomucoid solution (10-35 fig of ovomucoid). Changes in the percentage transmission were recorded. In a second type of determination, the order of addition of the ovomucoid and enzyme was reversed: An appropriate amount of the enzyme solution was added * Abbreviat,ions used: CM-cellulose, carboxymethyl cellulose; DEAE-cellulose, diethylaminoethyl cellulose; TAME, p-tosyl arginine methyl ester; BTEE, benzoyl-tyrosine ethyl ester; TCAacetone, trichloroacetic acid-acetone (1 volume of aqueous 0.1 Jf trichloroacetic acid + 2 volumes of acetone).
TURKEY
OVOMUCOIDS
65
to a mixture of substrate-indicator-buffer solution and the inhibitor and the changes in the percentage t,ransmission were recorded. Acetylation.3 Proteins w-ere acetylated essentially according to the method of Fraenkel-Conrat (8). Two hundred mg of enzyme or ovomucoid was dissolved in 2 ml of half-saturated solution of sodium acetate and cooled in an ice bat’h. To the cooled solution, 0.02 ml of acetic anhydride was added in five equal increments over a period of 1 hour. The mixture was dialyzed, centrifuged to remove insoluble material? and lyophilized. When enzymes, especially trypsin, were acetylated, the yield of products was only 507,. Purification by chromatography was as recently described (3). The degree of acetylatioii was determined by measuring the change in the number of free amino groups upon acetylation. The number of free amino groups was determined by the ninhydrin method (8) as standardized against the Van Slyke method with selected samples (3). SuccinyZation.4 This was done according to the method described by Habeeb et al. (9) and Buttkus et al. (10). A 20-fold excess of solid succinic anhydride was added to a 1.5 or 10% solution of ovomucoid in 57, sodium bicarbonate. The reagent was added at room temperature at intervals over a ZO-minute period, with const,ant stirring. The mixture was left standing another 20 minutes, after which it was dialyzed against water and then lyophilized. In these studies, the two concentrations of ovomucoid, 1.5 and lo%, were used to obtain low and high succinylated derivatives. The degree of succinylation was determined as for acetylation. Reaction with ethyl acetimidate hydrochloride.6 Amidination was done according to the procedure of Wofsy and Singer (11) by reacting the protein with ethyl acetimidate hydrochloride. The reagent 3 The degree of acetylation of the amino groups of the particular preparations used in these experiments were: trypsin, 7370; chymotrypsin, 66%; turkey ovomucoid, 707c. 4 As determined by the ninhydrin method (8), the amount of succinylation of amino groups was 157c for the low succinylated and 60% for the high succinylated preparations, respectively. j The amount of modification of amino groups by amidinat,ion was calculated by two methods. The ninhydrin method (8) gave a value of 48% for the decrease in amino groups; the automatic amino acid analyzer gave a value of 60% for the decrease in lysine. In the amino acid analysis, a new peak of a modified amino acid was located close to histidine, between histidine and arginine. The amount of material in the new peak, approximately account,ed for the loss in lysine.
SIMLOT
66
AND FEENEY
was prepared as described by McElvain and Nelson (12). Five ml of a lyO solution of ovomucoid in 0.1 111 borate buffer, pH 8.5, were mixed with 0.25 ml of a 0.1 111 solution of ethyl acetimidate hydrochloride in 2 III NaOH, and the pH was adjusted to 8.4. After the mixture was cooled, the pH was maintained in a pH stat for 2 hours. Then it was dialyzed and lyophilized. Zodination. Iodination was done as described by Stevens and Feeney (3) for a high level of iodination. A 10% solution of turkey ovomucoid at pH 8.5 was treated with iodine solution in potassium iodide. A sample of the iodinated ovomucoid was also acetylated with acetic anhydride as described earlier. RESULTS
Efects of chemical naod$cation of b-key ovomucoid. Table I summarizes the inhibitory activities of several differently modified ovomucoids. All three reagents which modify amino groups inactivated t,he trypsin inhibitory activity without affecting the chymotrypsin inhibitory activity. These reagents essentially caused no change in charge (amidination), a single change in
TABLE ACTIVITIES
I
OF CHEMICALLY TURKEY OVOMUCOID
Reagents
1. Acetic anhydridea 2. Succinic anhydrideb Low High 3. Ethyl acetimidate 4. Iodine in HI soln.a 5. Iodine + acetic anhydride”
MODIFIED
“rp Inhibitory actwity of contra: Trypsin
Chymotrypsin
<5
100
18 <5 <5
100
100 100 100
100 100
a In agreement with Stevens and Feeney (3). b Low succinylation was obtained with lCyO protein solutions and high succinylation with lO7C protein solutions. Experimental procedure is given in the text. c Ovomucoid was first iodinated with an iodine solution in KI in alkaline solution, dialyzed and lyophilized. The lyophilized iodinated ovomucoid was then acetylated with acetic anhydride.
gradual. The initial slopes of the plots depended also upon the ratio of enzyme to negative charge per amino group substituted ovomucoid. A low ratio gave sharper slopes (acetylation) or two changes in negative in the initial period, and vice versa. The rate charge per amino group substituted (suc- of interaction between chymotrypsin and cinylation). Iodination, which strongly inturkey ovomucoid was not affected, howcreasesthe charge, did not cause any losses ever, by the presence of four times the of activity while iodination combined with amount of trypsin as chymotrypsin. acetylat,ion inactivated only the inhibitory The rates of reaction of duck ovomucoid activity for trypsin. These results extend with trypsin and chymotrypsin were similar the previous findings of Stevens and Feeney to the rates of turkey ovomucoid, but (3) w&h carbamylat’ion, acetylation, or ovomucoid from the ring-necked pheasant iodination. They indicate that 6he change react,ed rapidly with both chymotrypsin in charge introduced by the modification is and trypsin. Chicken ovomucoid reacted not t#he cause of inactivation of the inhibirapidly with trypsin, and golden pheasant tory activit,y against trypsin. ovomucoid reacted rapidly with chymoRelative rates of yeaction with trypsin and trypsin. chymotrypsin. When the length of incubaConfirmatory estimations of the rates of tion of the mixtures of inhibit’or and enzyme hydrolysis of substrate by direct det’erminaprior to addition of substrate was varied, tion of the amounts of alkali consumed in trypsin and chymotrypsin reacted with avian the reaction were comparable, in general, to ovomucoids at different rates (Fig. 1). With the rates obtained by the spectrophottoincreased time of incubation, the residual metric method.6 enzymic activity decreased until maximal 6 The reaction as shown in Fig. 1 appears to be inhibition was reached. In general, this of second order, but the calculation of rate conoccurred considerably later with chymo- stants is difficult with this type of data. The orders trypsin than with trypsin. The activity of of the reactions and the calculations of rate contrypsin decreased rapidly, while the decrease stants are presently under investigation by more in activity of chymot’rypsin was more direct methods.
CHEMICALLY
MODIFIED
TURKEY
Tryp-Ovomumd
(18.10)
vowcold
60
120
TIME OF INCUBATION
180
240
OF ENZYME (seconds’
67
OVOMUCOTDS
(22:15)
300
360
420
AND INHIBITORS
FIG. 1. Times required for inhibition of trypsin and a-chymotrypsin by turkey ovomucoid. Numbers in parentheses refer to weight ratios of trypsin (Tryp) or chymotrypsin (Chymo) to turkey ovomucoid. Enzyme and ovomucoid were incubated together in pH 8.2 buffer for the periods indicat.ed, and then mixed with a solution containing substrate, indicator, and buffer. Changes in percent transmission at 395 mp were followed immediately on the recording spectrophotometer. Details are given in text.
Effects of acetylation of turkey ovomucoid on enzymes. In our earlier studies we showed that acetylation of turkey ovomucoid destroyed its capacity to inhibit trypsin but did not, destroy it,s capacity to inhibit a-chymotrypsin. This inactivation of the inhibitory activit,y for trypsin has been confirmed in the current studies. However, it, also has been found bhat the inhibitory activity for chymotrypsin was affected. The effect was expressed primarily as a 2-3fold increase in rate of inhibition (Fig. 2). This gave the illusion of a much greater comparative inhibitory activity of the acetylated ovomucoid for chymotrypsin if the determinations were performed without allowing sufficient time for the relatively slow reaction bet#ween the unmodified inhibitor and the enzyme to occur. A more
rapid reaction of the acetylated ovomucoid also was apparent in “delay t.ime” determinations (Fig. 3). A slightly increased total apparent inhibitory activity of the acetylated ovomucoid for chymotrypsin also was observed consistently. This apparent activity was approximately 5% greater than that of the unmodified ovomucoid on a stoichiometric basis, and was considered a reflection of higher affinity for the enzyme. Acetylated trypsin was essentially uninhibited by turkey ovomucoid, confirming similar findings with chicken ovomucoid (13). Acetylated chymot’rypsin was still inhibited by turkey ovomucoid, however, but the reaction was slow (Figs. 2 and 3). The apparent relative rates of interaction for acetylated turkey ovomucoid and acetylated chymotrypsin were in the following order (beginning with the most rapid):
GS
SIMLOT
I 60
I 120
TIME OF INCUBATION
AND
FEENEY
I 180
240
OF ENZYME (seconds)
300
I 420
360
AND INHIBITOR
FIG. 2. Effects of acetylation on times required for inhibition of ar-chymotrypsin by turkey ovomucoid. Weight ratios of chymotrypsin to turkey ovomucoid were 22:15. Abbreviations used: Chymo, chymotrypsin; AcChymo, acetylated chymotrypsin; TO, turkey ovomucoid; AcTO, acetylated turkey ovomucoid. Enzyme and ovomucoids were incubated together in pH 8.2 buffer for the periods indicated and then mixed with a solution of the substrate, indicators and buffers. The changes in percent transmission at 395 mp were then immediately followed on the recording spectrophotometer. Details are given in text.
acetylovomucoid-chymotrypsin; ovomucoidchymotrypsin; acetylovomucoid-acetyl-chymotrypsin; and ovomucoid-acetylchymotrypsin.6 Delay time assays. Green (7) described this technique for measuring displacement of the substrate, by the inhibitor, from the enzyme-substrate complex. In his experiments, the inhibitor was added to mixtures of the enzyme and substrate and the times required for inhibition to occur were noted. It is evident from Figs. 3 and 4 that there was a demonstrable time required for the occurrence of inhibition, confirming Green’s result,s with chicken ovomucoid. When the order of addition of the enzyme and ovomucoid was changed (Figs. 3 and 4), however, no significant’ differences were observed
in the times required for inhibition to occur. There thus was no evidence that the inhibitor displaced the substrate from the enzyme-substrate complex. The delay time assays also were used to study the effects of modification on the relative rates of interaction with the enzymes (Figs. 3 and 4). All three modifications of ovomucoid directed at the amino groups inactivated the antitryptic activity, as also was found by the direct methods (Fig. 1; Table I). However, major increases were found in rates of inhibitions of iodinated derivatives with both trypsin and and amidination did not chymotrypsin, cause an increased rate with chymotrypsin as did acetylation or succinylation. All three modifications
causing
an
increased
negative
CHEMICALLY
MODIFIED
TURKEY
charge, t’herefore, increased the rate of interactions, while the modification causing no significant change in charge (amidination) did not increase hhe rat,e.
OVOMUCOIDS
69
CHYMOTRYPSIN
CHYMOTRY PSIN
AC Chy -TO AC Chy - Ac T O
Chy -TO
so-
1 I I I Po,nt of Add,t,on of Ovomucoid I
Chy -AC T O
I 120
I 240 SECONDS
I 350
I 480
I 360
I 480
80 -
120 SECONDS
55
240
360
TRY PSIN t
AC Try -AC T O AcTry-TO
Try -TO
Potnf of Addltoan I 120
of Ovamuco,d I 240
I
SECONDS
120 SECONDS
240
360
FIG. 3. Times required for inhibition of tryspin and a-chymotrypsin by turkey ovomucoid with “delay time” technique. Abbreviations used: Chy, chymotrypsin; AcChy, acetylated chymotrypsin; TO, turkey ovomucoid; AcTO, acetylated t,urkey ovomucoid; Try, trypsin; AcTry, acetylated trypsin. Weight ratios were 22:15 for chymotrypsin and ovomucoid and 18:15 for trypsin and ovomucoid. Ovomucoid, buffer, indicator and substrate were mixed together and then the enzyme added. The acetylated chymotrypsin had a specific activit.y nearly identical to the original chymotrypsin. acetylated trypsin had a slightly loaer activity than the original trypsin and the plot for acetylated trypsin was similar to the plot given for AcTry-AcTO. Details are given in text.
FIG. 4. Effect of chemical modification on delay time assays. Abbreviations used: Chy, chymotrypsin; TO, turkey ovomucoid; AcTO, acetylated turkey ovomucoid; Try, trypsin; AmTO, amidinated turkey ovomucoid; SucTO, succinylated turkey ovomucoid; ITO, iodinated turkey ovomucoid; IAcTO, iodinated and acetylated turkey ovomucoid. To a mixture of enzyme-buffer and substrate, was added turkey ovomucoid solution within 18-25 seconds and the inhibition recorded on a chart at 395 rnp. The weight ratios were: (a) for chymotrypsin and turkey ovomucoids 22:15; (b) for trypsin and turkey ovomucoids 18:15.
DISCUSSION
Several previous studies have concerned both the mcehanism of inhibition by inhibitors of trypsin and the kinetics of the inhibitory process. Avian ovomucoids have been shown to form physically demonstrable
70
SIMLOT
AND
complexes (1, 2, 14). It has been usually accepted that the mechanism of inhibition involves the formation of a relatively &able complex (15). Early studies with chicken ovomucoid and trypsin, with casein as a substrate, indicated that the inhibition was of t,he “noncompetitive” type (13), but later studies with synthetic substrate were interpreted as showing that a variety of natural inhibitors of trypsin were competitive inhibitors (7, 16, 17). Comparative studies also showed that the pancreatic trypsin inhibitors reacted with trypsin much more slowly than did ot’her inhibitors, including chicken ovomucoid (7). It has been suggest.ed that electrostatic forces are important in the formation of complexes between trypsin and its natural inhibitors (15). In most instances rather large differences were found between the isoelectric point of bhe inhibitor and that of the enzyme. In only one instance mere the isoelectric points close together. This occurred with trypsin and the pancreatic t#rypsin inhibitor, and the reaction between them was relat,ively slow (17). In more recent studies the interaction of trypsin with trypsin inhibitors was found t’o be accompanied by a release of a titrat>able proton (18). Finkenstadt and Laskowski, Jr. (19) have presented evidence for the formation of a covalent intermediate and possible enzymatic hydrolysis of a peptide bond. In the present studies we have found still further differences between t.he properties of avian ovomucoids t’han we have reported previously. In addition to the inactivation of the trypsin-inhibitory activity of turkey ovomucoid by acetylation or carbamylation (without affecting the chymotrypsin inhibitory activity) (3), both amidination and succinylation also were found to produce similar effects. Change in charge due to the modification, therefore, does not appear to be the cause of inactivation. Two other major differences were observed in the dual inhibitory properties of turkey ovomucoid against trypsin and chymotrypsin. One was a large difference between the rates of inhibition of the two enzymes by the ovomucoids. This was of such magnit.ude that, in actual laboratory determination of inhibitory activity by incubating
FEENEY
different mixtures of the enzymes and the inhibitor, an incubation time of only 1 minute usually was satisfactory with trypsin, while at least 3-4 minutes were required with chymotrypsin. The second difference was the effect’ of chemical modification of the ovomucoid on the rat,es of inhibit’ion of the enzymes. Acet’ylation or succinylation increased t.he rat,e of inhibition of chymotrypsin, while amidination did not increase the rate. In addition, iodination, which has no significant effect on the stoichiometry of inhibition of either trypsin or chymotrypsin by turkey ovomucoid (3), also increased the rates of reaction wit,h both trypsin and chymotrypsin. Our studies on relative rates indicate that factors related to charge are important in the interaction of ovomucoids with trypsin or chymotrypsin. However, observat8ions of the rat.es under different, conditions and the inactivation of t.he t,rypsin inhibitory activity by reagents for amino groups which both do and do not affect charge, prove conclusively that ot’her factors are also important. The interactions are thus cornplex and involve a variety of factors, including possibly such general ones like electrostatic and conformational factors and more specific ones like interactions with specific amino acid side chains. The inactivations of the t’rypsin inhibitory activity wit’h reagents for amino groups mdicate a specific involvement of one or more of the E-amino groups of lysine in t’he interaction with trypsin. Such a specific involvement has been suggested by Finkellst’adt and Laskowski (19) for t,he interactions of chicken ovomucoid with Drypsin. These authors have suggested that a covalent linkage is formed with the possible specific hydrolysis at an arginine bond (20). The complexities of the reactions as well as the effects of chemical modifications are so extensive, however, that many st,udies are necessary. For example, it is highly probable that some particular chemical modificat,ion might have opposing effects which might therefore either enhance or decrease the activity depending upon the relative importance of these effects. Since the order of addition of the enzyme and inhibitor in the delay-time studies was
CHEMICAL
MODIFIED
not significant and the inhibitor does not influence deacylation reaction,’ it appears that the procedure measures the rate of association of free enzyme and inhibitor and not the displacement of the substrate by the inhibitor, as suggestedby Green (7). A conventional scheme for the enzymic sequence might be:
in which E, S, ES, and ES* are the enzyme, substrate, initial enzyme-substrate complex, and enzyme-modified substrate complex, respectively; and PI and Pz are the products of hydrolysis of the substrate. According to our interpretation, the inhibitor would form a complex with the free enzyme and the rate of such formation would be much slower than any of the reaction rates between the enzyme and substrate. The formation of a complex between the inhibitor and enzyme, therefore, should influence the course of the reaction between the enzyme and substrate only by relatively slowly removing enzyme from the system. .XKNOWLEDGMENT Appreciation C. Stevens for servations on ovomucoid. 7 In of the ported rate of
is due David T. Osuga and Frits advice and for certain of the obthe properties of acetyl turkey
a personal University that turkey deacylation
communication, Dr. John Mehl of Southern California has reovomucoid does not affect the of cinnamoylchymotrypsin.
TURKEY
OVOMUCOIDS
71
REFERENCES 1. FEENEY, R. E., STEVENS, F. C., AND OSUGA, D. T., J. Biol. Chem. 238, 1415 (1963). 2. RHODES, M. B., BENNETT, N., AND FEENEY, R. E., J. Biol. Chem. 236, 1686 (1960). 3. STEVENS, F. C., AND FEENEY, R. E., Biochemistry 2, 1346 (1963). 4. SCHONBAUM, G. R., ZERNER, B., AND BENDER, M. L., J. Biol. Chem. 236,293O (1961). 5. LINEWEAVER, H., AND MURRAY, C. W., J. Biol. Chem. 171, 565 (1947). 6. RHODES, M. B., HILL, R. M., AND FEENET, R. E., Bnal. Chem. 29, 376 (1957). 7. GREEN, N. M., J. Biol. Chem. 206, 535 (1953). 8. FRAENKEL-CONRAT, H., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. IV, p. 247. Academic Press, New York (1957). 9. HABEEB, A. F. S. A., CASSIDY, H. G., AND SINGER, S. J., Biochem. Biophys. Acta 29, 587 (1958). 10. BUTTKUS, H., CLARK, J. R., AND FEENEY, R. E., Biochemistry 4, 998 (1965). 11. WOFSY, L., AND SINGER, S. J., Biochemistry 2, 104 (1963). 12. MCELVAIN, S. M., AND NELSON, J. W., J. Am. Chem. Sot. 64, 1825 (1942). 13. FRAENKEL-CONRAT, H., BEAN, R. S., AND LINEWEAVER, H., J. Biol. Chem. 177, 385 (1949). 14. RAM, J. S., TERMINIELLO, L., BIER, M., AND NORD, F. F., Arch. Biochem. Biophys. 62, 451 (1954). 15. LASKOWSKI, M., AND LASKOWSKI, Jn., M., Advan. Protein Chem. 9, 203 (1954). 16. GREEN, N. M., AND WORK, E., Biochem. J. 54, 347 (1953). 17. GREEN, N. M., Biochem. J. 66,407 (1957). 18. LEBOWITZ, J., AND LASKOWSKI, JR., M., Biochemistry 1, 1044 (1962). 19. FINKENSTADT, W. R., AND LASKOWSKI, JR., M., J. Biol. Chem. 240, PC962 (1965). 20. FINKENSTADT, W. R., OZAWA,K., AND Las~owSKI, JR., M., Federation Proc. 24,593 (1965).