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deuterium atom on the methylene carbon of the side chain is indicated by the collapse of the methyl triplet to a doublet at 1.7 and by a decrease in the integral for the broad multiplet at 3.1. Thus, a deuterium atom shifted from the tertiary carbon to the methylene carbon of the ethyl side chain in the course of formation of 2. The E~(DPM)~-shifted nmr spectra of the normal hydroformylation products 4-d and the diastereomeric mixture 5-d both contained singlets for the 4-methyl and the 3-methyl groups, respectively, and demonstrated that these products were formed without shift of deuterium.
nature of the binding of the copper in the hemocyanins from the two different species, appear to be borne out by certain distinctive features in their respective circular dichroism (CD) spectra as described below. The hemocyanins employed in these experiments (obtained from the Marine Biological Laboratories, Woods Hole, Mass.) were usually used as naturally present in the hemolymph (serum) of the animals. However, before use, the hemocyanin hemolymph was conditioned4 so as to remove extraneous clotting protein. The hemocyanin hemolymph was diluted with 0.05 M , pH 7.0, sodium dihydrogen phosphate buffer. The oxygen-carrying capacity was measured Charles P. Casey,* Clifford R. Cyr by the absorbance ( A , ) at 345 nm and the activity of the Department of Chemistry, University of Wisconsin hemocyanin was expressed as the per cent oxygenMadison, Wisconsin 53706 carrying capacity, defined as the ratio of the A . of Receiaed December 17, 1970 irradiated hemocyanin to that of a nonirradiated control sample. Deoxygenation of oxyhemocyanin was accomplished by passage of helium through the hemoInactivation and Reactivation of Hemocyanin cyanin s o l u t i ~ n . The ~ ~ ~total copper content of hemoby Radiolytic OH and e,, cyanin was determined by atomic absorption. The Sir: cuprous copper fraction in deoxyhemocyanin was When exposed to ionizing radiation the biochemical measured by the 2,2’-biquinoline method.’ The C D function of enzymes and proteins can be impaired or spectra were recorded with a Cary-60 spectropolardestroyed. In this communication we demonstrate imeter on samples of hemocyanin isolated and purified that ionizing radiation can also restore the biochemical by ultracentrifugation and dialysis. Irradiations were function of a previously radiation-damaged macrocarried out at 25 f 1O with a cobalt-60 y source. molecule. Hemocyanin from Busycon was first inactivated by irIn experiments with the oxygen-carrying copper radiation in oxygenated media with 17 krads of cobalt-60 protein, hemocyanin, we find that in neutral, oxygeny radiation followed by deoxygenation as described free media, radiolytically produced OH eliminates the prevjously. 3 , 4 Immediately afterward, in an attempt oxygen-carrying capacity by oxidizing the proteinto restore the oxygen-carrying capacity, the inactivated bound copper to the cupric state, while the primary hemocyanin was irradiated with varying doses in reducing species eaq- fully restores the oxygen-carrying oxygen-free media, in the presence and absence of 0.5 M capacity by reducing cupric copper to the cuprous state. sodium formate, an OH radical scavengers8 In the Similarly, the secondary radical 1 , 2 COz-, produced when absence of formate, no restoration of the oxygenformate is used as an OH scavenger, is capable of carrying capacity was observed (Figure 1). However, restoring inactivated hemocyanin. The radiation doses in the presence of formate the oxygen-carrying capacity employed (0-110 krads) do not cause any significant increased with increasing dose to the point that at 60 changes or damage to the protein moiety. It has been krads practically all of the original activity was restored. demonstrated p r e v i o ~ s l ythat ~ ~ ~the elimination and Concomitantly, the cuprous ion concentration inrestoration of oxygen-carrying capacity of hemocyanins creased in the same manner as the oxygen-carrying by irradiation in oxygenated media was solely due capacity (Figure 1). The restored or reactivated to HzOzproduced during irradiation. It has long been hemocyanin appeared to be identical with that of the known that HzOz and other oxidizing-reducing agents nonirradiated hemocyanin, e.g., the oxygenationmodify the oxygen-carrying capacity of h e m o ~ y a n i n . ~ , ~deoxygenation cycles, optical absorption spectra, and We also find that the reduction of Cu(I1) to Cu(1) C D spectra. parallels the restoration of the oxygen-carrying capacity Since the secondary COz- radical is capable of of hemocyanin derived from one species of Mollusca reducing the cupric ion, we investigated its restorative (Busycon, the channeled whelk). However, no restoability in the absence of e,, by irradiating inactivated ration of oxygen-carrying capacity is effected in hemoBusycon hemocyanin in the presence of both NzO, an cyanin obtained from a species of Arthropoda (Limulus, efficient eaq- scavenger, and sodium formate. We the horseshoe or king crab), despite the fact that comfound that the COz- radical did, indeed, reactivate plete reduction of Cu(I1) to Cu(1) occurs following Busycon hemocyanin. Finally, we investigated the irradiation in the presence of suitable scavengers. restorative capability of eaq- in the absence of the COSThese observations, indicative of a difference in the radical by carrying out irradiations in the presence of 2-propanol. Again, we observed restoration of Busycon (1) D. M. Donaldson and N. Miller, Radiar. Res., 9, 487 (1958); hemocyanin. N. Miller, ibid., 9,633 (1958). In related experiments with Limulus hemocyanin, (2) A. Fojtik, G. Czapski, and A. Henglein, J. Phys. Chem., 74, 3204 ( 1970). we found that neither eaq- nor the COZ- radical restored (3) J. Schubert and E. R. White, Science, 155, 1000 (1967). the oxygen-carrying capacity. However, the reduction (4) J. Schubert, E. R . White, and L. F. Becker, Jr., Adcan. Chem. Ser., No. 81, 480 (1968). of Cu(I1) to Cu(1) increased with increasing radiation ( 5 ) G. Felsenfeld and M. P. Printz, J. Amer. Chem. Soc., 81, 6259 (1959). (6) R. Lontie and R. Witters in “The Biochemistry of Copper,” J.
Peisach, P. Aisen, and W. E. Blumberg, Ed., Academic Press, New York, N. Y., 1966, pp 455-463,
Journal of the American Chemical Society
(7) G. Felsenfeld, Arch. Biochem. Biophys., 87,247 (1960). (8) E. J. Hart, J. I
