COMMUNICATIONS TO TIIE EDITOR
26.56
hibitory action of a proton is reinforced by the dicarboxylic acid. Under certain conditions even the keto acid substrates may act as inhibitors rather than activators of the transamination.8 The spectral changes with pH may be interpreted as follows. In terms of the studies of Metzler and S r ~ e l l the ~ . ~ forms of the pyridoxal imine which may affect the spectra nrr shown in Fig. 3.
Vol. 79
ammoniacal buffer at pH 9.2. Figure 1 shows current voltage curves of copper(T1) a t the rot.itciI platinum electrode (RPE)in a 1)uffcr 0 1 1 1 111 . I : [ : monk and 0.1 dVin animoniurn riitr'tte (curve 13) The first wave corresponds to the reduction o f copper(1I) to copper(1). Yo copper wave5 are 01) served when albumin is present in a 1iio1:tr concuitration greater than t h a t of copper (curve 1)) Curve E illustrates the reappearnnce of thc coppc r waves when n n excess of copper is present.
I"
t
1
0.2 u.l U.0 0,s i.0 Potential vs. S.C.E., v. Fig. 1.-Current voltage curves at rotated platinum elcctrode; buffer, 0.1 M NH3 -t 0.1 M r\W,NOJ; pI1 9.2; speed of rotation, 900 r.p.m.: A, residual; B, 4.0 X 1 0 F .If Cu(I1); C, 7 . 3 X 10-6 ]If BSA; D, 7 . 3 X Af BSA i6 . 5 x io-6-w C U ( I I ) : E, 7.3 x 10-5 M BSA 13.0 x 10-6 111 Cu(1I). 0
A Fig. 2.-The
+
ionization of pyridoxal phosphate imines
By amperometric titration a t the RPE in ammonia buffer a t -0.4 volt, we have found that copper(I1) reacts rapidly with native 13s-1in a mole ratio of one to one. Similar results have been ohtained under proper conditions at pH S and 10. The rapid reaction is followed by a slower reaction of additional amounts of copper(I1) ; this does not interfere with the titration. The sulfhydryl group is not involved in the reaction with copper(T1). After addition of one or two moIes of copper(I1) per mole BSA, 0.6s niole sulfhydryl per mole BSA was found by subsequent ainperometric titration with mercuric chloride andlor silwr nitrate.? Also, addition of O.GS mole of si1vc.r nitrate or mercuric chloride per mole BSA prior to titration with copper(I1) did not affect the results (8) Cf. P. Peyser, Doctoral Dissertation, Columbia University, of this titration. New York, N. Y., 1954. Titrations of BSA have also been carried out in a (9) D . E. Metzler and E. E. Snell, THISJ O U R N A L , 7 7 , 2431 (1955). denaturing mixture which was 4 M in guanidine DEPARTMENT O F BIOLOGY in anihydrochloride, 0.1 116in ammonia and 0.1 MASS. INST. OF TECHNOLOGY W. TERRY JENKINS 39, Mass. CAMBRIDGE IRWINW.SIZER monium nitrate. A reaction ratio of copper(I1) to BSA somewhat greater than unity (about 1.3) RECEIVEDMARCH21, 1957 was found. This higher reaction ratio is ascribed to oxidation by ~ o p p e r ( 1 1of) ~the sulfhydryl group A STABLE EQUIMOLAR COPPER(II)-ALBUMIN COM- which is oxidizable to disulfide in denatured 211-
These equations are based on the assumption that the yellow color may be ascribed to the hydrogen chelate ring. Rupture of this ring by a change in PH results in loss of color. A ~ R ofA6.2 for this color change for the transaminase suggests that it is the dipolar form of the imine which occurs at neutrality, in contrast to the non-polar forms of the B 5.9, ~ K = D imines studied by ;LletzlerR( ~ K = 10.5). The implications of these findings with reference to the mechanism of enzymatic transamination will be discussed elsewhere. Acknowledgment.-We are happy to acknowledge financial support from Ethicon, Inc.
PLEX
Sir : A simple and rapid amperonictric titration technique has been developed for the determination of bovine serum albumin (BSA) with copper(I1) in
(1) W.I..Hughes, Jr., THISJ O U R N A L , 69, 1830 (1947); Cold Spriits Harbor Symposium Q I L Q ~ ~Biol., . 14, 79 (1949). ( 2 ) I. M. Kolthoff. W. Stricks and L. hlorren, Anal. C h e m . , 26, 300 (1951): I. hf. Kolthoff and W. Stricks, TnIs JOWRXAL, 72, 1952 (1950). (3) I.
IT. Kolthoff and W. S t r i c k s , . A m i . fhsnz., 23, 763 (1951).
May 20, 1957
COMMUNICATIONS TO THE EDITOR
2657
bumin. When the sulfhydryl group in denaturing The sequence of a portion of the active site of medium is inactivated by addition of 0.68 mole of chymotrypsin has been e ~ t a b l i s h e d ~and - ~ hence a silver nitrate or potassium ferricyanide, the reac- comparison could be made if similar information tion ratio of copper(I1) .to BSA was again found to could be obtained for an enzyme of very different be unity. specificity. Such an opportunity arose when it was Our experiments have been carried out under demonstrated that the active site of phosphoglucoconditions quite different from those of Klotz, mutase was marked by a serine pho~phate.’,~ et a1.,4 and no comparison with their results is made P32-labeled enzyme was prepared by exchange here. Our results show that in the pH range with radioactive substrates ( g l ~ c o s e - l - P O 4and ~~ 8-10, one copper(I1) is bound to the BSA molecule g l ~ c o s e - 6 - P O ~ under ~ ~ ) conditions similar to those extremely tightly, and that the sulfhydryl group is of the standard assay.g I t had been established not the reactive group. It is of interest to note that the enzyme is labeled only a t the active site that nickel(II), even in very small concentrations, by this procedure.8 The enzyme was then deand also, to a lesser extent, cobalt(I1) greatly inter- graded, either with acid alone, with proteolytic fere with the copper(I1) BSA reaction while even enzymes alone, or with proteolytic enzymes follarge amounts of zinc(I1) have very little effect. lowed by acid hydrolysis. Radioactive phosphoThus, the group in the BSA molecule responsible peptides were isolated by use of Dowex 50 colfor the binding of copper(II), the nature of which umnsl’J.ll and paper chromatography. The comis a t present unknown, apparently also forms stable positions of the phosphopeptides are shown in complexes with nickel(I1) and cobalt(I1) but not Table I. with zinc(I1). One unique site in the BSA moleTABLE I cule is the N-terminal aspartyl group, and further P a 2 P ISOLATED ~ ~ FROM ~ PHOSPHOGLUCO~ ~ ~ ~ ~ work may show whether this group is the site of MUTASE copper(I1) binding. Phosphopeptide There is very little (if any) reaction between Pepobtained by tide treatment with Amino acid compn. copper(I1) and human 7-globulin under our conditions. The method has been applied to the deter1 Proteolytic acid (Asp,Ser,Gly,Glu) mination of albumin in blood serum, and prelim2 Proteolytic acid (Asp,Ser,Gly,Glu,Ala) inary results are promising. 3 Proteolytic acid (Asp,Ser,Gly,Glu,Ala,Val,Thr) 4 Proteolytic only (Asp,Ser,Gly,Glu) Acknowledgment.-This work was supported by 5 Proteolytic only (Asp,Ser,Gly,Glu Ala,Val,Thr, grants from the U. S. Public Health Service and Leu) from the Louis and Maud Hill Family Foundation.
+ + +
(4) I. M. Klotz and H. G. Curme, THISJOURNAL, 70, 939 (1948); H.A. Fiess and I. M. Klotz, ibid., 74, 887 (1952); I. M. Klotz. J. M. Urquhart and H. A. Fiess, ibid., 74, 5537 (1952); I. M .Klotz, J. M Urquhart, T. A. Klotz and J. Ayers, ibid., 77, 1919 (1955); I. M. Klotz, I. L. Faller and J. M. Urquhart, J . Phys. Colloid Chem., 64, 18 (1950); I. M. Klotz and H. A. Fiess, ibid., 66, 101 (1951).
6 7
Acid only Acid only
(Asp,Ser,Gly,Glu,Ala) (Asp,Ser,Gly,Glu,Ala,Val Thr, Leu)
While the detailed sequence has not been completely established, it is to be noted that the com(5) On leave of absence from Bucknell University, Lewisburg, Pa. position of each peptide and the order from comSCHOOL OF CHEMISTRY parison of lower and higher peptides is in perfect OF MINNESOTA UXIVERSITY I. hl. KOLTHOFF h!fINNEAPOLIS 14,MISN. B. R. WILLEFORD,J R . ~ agreement with the sequence for chymotrypsin which is Asp Ser Gly Glu Ala Val.4-6 The probRECEIVED MARCH25, 1957 ability that the 7 peptides isolated from phosphoglucomutase would agree with this sequence ENZYME CATALYSIS AND ENZYME SPECIFICITYby chance alone is very small. Moreover the conCOMBINATION O F AMINO ACIDS AT T H E ACTIVE cordance of the acid derived and proteolytic enSITE OF PHOSPHOGLUCOMUTASE1 zyme plus acid derived peptides is strong evidence Sir : against the sequence being an artifact of the sepaIn the classical template model for enzyme action ration procedure. The data therefore indicate that the substrate is assumed to be absorbed with a pre- the amino acid sequence of the active site for a t cise fit into an area called the “active site” of the least 6 amino acids is the same for an enzyme which protein.2 Presumably the amino acids a t this site specifically hydrolyzes peptide bonds and an enprovide not only the catalytic action but also the zyme that specifically transfers phosphate bespecificity of the enzyme. From the widely varying tween carbohydrate molecules. specificity patterns of enzymes it would be anticiThis apparently surprising result is good suppated that the amino acid composition a t the port for the above mentioned hypothesis that comactive site would vary to fit the particular sub- mon bond-breaking mechanisms exist despite varystrate. On the other hand, stereochemical and (4) J. A. Cohen, R. A. Oosterbann, M. G. Warringa and H. S. other evidence had led to the hypothesis that a few Jansz, Disc. F a r a d a y Soc., 20, 114 (1955). bond-breaking mechanisms might be common to (5) N. K.Schaffer, S. Harshman, R. R. Engle and R. W. Drisko, reactions of widely different specificities3 and hence Federation Proc., 14, 275 (1955). (6) F. Turba and G. Gundlach, Bid. Z., 327, 186 (1965). there might be some common amino acid sequences. (7) G.R. Jolles and L. Anderson, Am. Chem. SOC.Abstracts, Sept.
(1) Research carried out at Brookhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission. ( 2 ) E. Fischer, Ber., 27, 2985 (1894). (3) D. E. Koshland, Jr., “Mechanism of Enzyme Action,” edited by McElroy and Glass, Johns Hopkins Press, Baltimore, Md., 1954, p. 608.
1955, p. 232. (8) E. P. Kennedy and D . E. Koshland, Jr., J. Bid. Chem., in press. (9) V. A. Wajjar, ibid., 176, 280 (1948). (10) M.Flavin, ibid., 210, 771 (1954). (11) N. K. Schaffer, S. Harshman and R. R. Engle, ibid., 214, 799 (1955).
~
~
