Studies on the substrate specificity of ceruloplasmin

528

PRELIMINARY NOTES

PN 1136

Studies on the substrate specificity of ceruloplasmin The enzymic properties of the serum copper-containing protein, ceruloplasmin, were first described b y HOLMBERG AND LAURELL1 over 20 years ago. These investigators showed that ceruloplasmin rapidly oxidized p-phenylenediamine, as well as DOPA, hydroquinone, catechol and epinephrine, although the latter group at a somewhat slower rate. Since that time additional substrates have been reported such as 5h y d r o x y t r y p t a m i n e 2 and other hydroxyindoles 3, and ascorbic acid4, 5 although there is considerable doubt concerning the last-named substance 6. However, little is known concerning relative reaction rates or structure-activity relationships, and the physiological implications of its oxidase activity are as yet obscure. The study now in progress shows that the oxidation of a large number of aryl polyamines and polyphenols is catalyzed b y ceruloplasmin and that the rates of 02 uptake are directly related to the HAMMETT sigma values7, 8 of these compounds. Ceruloplasmin was prepared from pig serum according to the method of HOLMBERG AND LAURELL9, and then purified on a DEAE-cellulose column as described by CURZON AND VALLET10. The resulting deep-blue solution, as well as all buffer solutions, were passed through a Dowex A-I Chelating Resin cohlmn to remove all traces of free copper 6. 02 uptake was measured in a Warburg apparatus at 37 °. Reactions were run in o.I M sodium acetate buffer (pH 5.5). All reagents used were of the highest purity available and were recrystallized or resublimed if necessary. For each substrate, the m a x i m u m velocity (Vmax) was determined according to the method of LINEWEAVER AND BURK11. The experimental results are summarized in Fig. I. For each substrate, Vmax, expressed as /~10~ consumed per h per fig ceruloplasmin Cu, is plotted semilogarithmically against the sum of HAMMETT sigma values for all ring substituents.

10 ~-

PPD® MPPD #DPP PAP. TAB •

8O~

HQ •

DMP*

_2. l o 2 .=

MPD,

8 10' O

I

I

I

I

I

-0.2 -0.4 -0.6 -0.8 -1.0 Sum of h a m m e t t sigma vQlues

1

-1.2

-1.4

Fig. i. PPD, p-phenylenediamine; MPPD, N-methyl-p-phenylenediamine; PAP, p-aminophenol; DPP, N,N-dimethyl-p-phenylenediamine; HQ, hydroquinone; TAB, triaminobenzene; DMP, N,N-dimethyl-m-phenylenediamine; MPD, rn-phenylenediarnine. Biochim. Biophys. Acta, 63 (~962) 528-529

PRELIMINARY NOTES

529

The sigma values were obtained from JAFFETMand HAMMETTis. The substrates include amines, phenols and phenolic amines. It is apparent that the more reactive compounds are those with the more highly negative sigma values. Thus, a reaction rate of 835 was obtained for p-phenylenediamine with a total sigma value of --1.32o, while m-phenylenediamine, with a total sigma value of --0.322 gave a reaction rate of only 39. Compounds having substituents with high positive sigma values showed little or no activity. Although o-phenylenediamine was readily oxidized, 4-nitro-o-phenylenediamine did not react. Similarly, the reaction rate with hydroquinone was 285, while 2,5-dihydroxybenzoic acid, the carboxyl analogue, showed only very slight activity. Both the nitro and the carboxyl groups possess high positive sigma values, large enough to offset the negative contributions of other ring substituents. A negative sigma value for an aryl substituent indicates that electrons are supplied to the ring by that group. A positive sigma value indicates the opposite effect. As a result the electron density of the ring is greater in those compounds having negativesigma-value substituents. It is evident, then, that an increased ring electron density in the substrate is associated with increased activity with ceruloplasmin. A second point involves formation of the substrate-enzyme complex. Since ceruloplasmin will not attack aromatic monoamines or monophenols, one might consider that the points of attachment would be at the ring substituents, such as the two amino groups in p-phenylenediamine. However, it has also been found that compounds of this type which are fully substituted (e.g. N,N,N',N'-tetramethylp-phenylenediamine) are readily oxidized by ceruloplasmin. Therefore, it is suggested that enzyme binding may not be through the amine or phenolic groups, but instead directly to the ring, or more specifically, to the rr electrons of the ring. We wish to thank Miss J. LENNANE for skillful technical assistance. This work was supported in part by a grant from the U.S. Public Health Service, National Institutes of Health (Grant No. H-645o ).

Departments of Pharmacology and Biochemistry, Albert Einstein College of Medicine, Yeshiva University, New York, N.Y. (U.S.A.)

WALTER G. LEVlNE* J. PEISACH**

1 C. G. HOLMBERG AND C. B. LAURELL, Acta Chem. Scand., 5 (1951) 476. C. C. PORTER, D. C. TiTus, B. E. SANDERS AND E. V. C. SMITH, Science, 126 (1957) lO14. 3 H. BLASCHKO AND W. LEVlNE, Brit. J. Pharmacol., 15 (196o) 625. 4 F. L. HUMOLLER, M. P. MOCI~LER, J. M. ]-IOLTHAUS AND D. J. MAHLER, J. Lab. Clin. Med., 56 (196o) 222. 5 C, WALTER AND E. FEIEDEN, Federation Proc., 21 (1962) 246. s A. G. MORELL, P. AISEN AND I. H. SCHEINBERG, personal communication. L. P. HAMMETT, J. Am. Chem. Soc., 59 (1937) 96. s L. P. HAMMETT, Trans. Faraday Sot., 34 (1938) 156. 9 C. G. HOLMBERG AND C. B. LAURELL, Acta Chem. Scand., 2 (1948) 55o. 10 G. CURZOl~ AND L. VALLET, Biochem. J., 74 (196o) 279. 11 H. LINEWEAVER AND D. J. BURK, J. Am. Chem. Soc., 56 (1934) 658. 1~ H. H. JAFEE, Chem. Rev., 53 (1953) 191. la L. P. HAMMETT, Physical Organic Chemistry, McGraw Hill, New York, 194 o, p. 188.

Received July i2th, 1962 " Recipient of U.S. Public H e a l t h Service Research Career D e v e l o p m e n t Award GM-K 381 o2-C1-A. "* P r e s e n t address: D e p a r t m e n t of Chemistry, Yeshiva University, New York 33, N,Y. (U.S.A.).

Biochim. Biophys. Acta, 63 (1962) 528-529