Howard S. Mason

Melanoproteins. I. Reactions between enzyme-generated quinones and amino acids.

Abstract 1. 1. A systematic survey of the reactions of compounds containing the functional groups of proteins with melanogenic systems comprised of o-diphenol oxidase and catechol, 4-methylcatechol, 3,4-dihydroxyphenylalanine, or 5,6-dihydroxyindole, has been carried out with an automatic recording rapid-scanning spectrophotometer. 2. 2. The 3-substituted indole ring, the amide group, the ureido and guanidino groupings, seryl hydroxyl, and 4-substituted imidazole groups do not react with any of the quinones derived by enzymic oxidation of the catecholic melanogens, according to spectroscopic criteria. 3. 3. N-terminal primary amino groups, aliphatic amino groups, secondary amines in amino acids, and thiol-containing amino acids react with o-benzoquinone and 4-methyl-o-benzoquinone to give intermediates with characteristic absorption spectra. 4. 4. Only thiol-containing compounds (and aromatic amines) react with melanogenic quinones derived from 3,4-dihydroxyphenylalanine to produce intermediates with characteristic absorption spectra. These reactions appear to involve “indole-5,6-quinone” only. 5. 5. A new melanochrome or leucomelanochrome which forms during the polymerization of indole-5,6-quinone to melanin, has been observed.

Hydroxylation catalyzed by peroxidase

Abstract 1. 1. The influence of substituents upon the orientation of aromatic hydroxylation by the dihydroxyfumarate-peroxidase-oxygen and the ascorbate-iron-oxygen systems, has been studied. 2. 2. Phenolic hydroxyl directs the entering hydroxyl group to the ortho and para positions in both systems. 3. 3. The nitro group directs the entering hydroxyl to all positions in the dihydroxyfumarate system; m-nitrophenol is formed in preponderance. 4. 4. The yield of phenolic products from the hydroxylation of nitrobenzene, benzole acid, and salicylic acid under comparable conditions is 1.45, 8.95, and 9.40 μmoles, respectively. The relative yields appear to be more characteristic of free radical attack than electrophilic substitution. 5. 5. Hydrogen peroxide does not substitute for oxygen in the dihydroxyfumarateperoxidase system, and manganese inhibits hydroxylation. These and other properties are explained by the assumption that the hydroxylating agent is perhydroxyl or a related radical, formed in accordance with the Yamazaki peroxidase-oxidase mechanism.

An electron-spin resonance study of coenzyme B12☆

The absence of electron-spin resonance spectra from three different samples of coenzyme B12 either in the solid crystalline state or in aqueous solution suggests that coenzyme B12 is diamagnetic. These results agree with several magnetic susceptibility measurements and with the chemical degradation studies; they are, however, in disagreement with other magnetic susceptibility measurements which indicate that coenzyme B12 is paramagnetic. In contrast, electron-spin resonance spectra of coenzyme B12 photolyzed under anaerobic condition indicate that the photolysis product is paramagnetic. Since this spectrum is identical to that obtained for vitamin B12r, it has been concluded that the anaerobic photolysis product contains bivalent cobalt. Furthermore, exposure of the anaerobic photolysis product or vitamin B12r to oxygen completely abolishes the electron-spin resonance spectra. This observation is in accord with the magnetic susceptibility studies of aquocobalamin, known to be the product of this oxidation reaction. These results are interpreted to mean that coenzyme B12 does not contain bivalent cobalt. Consequently, the photolytic decomposition of coenzyme B12 must involve an intramolecular oxidation reduction or a homolytic cleavage of the carbon-cobalt linkage. The electron-spin resonance spectra do not establish the valence state of cobalt in coenzyme B12; however, the absence of a signal suggests that coenzyme B12 is diamagnetic and consequently contains trivalent cobalt.

Dimorphism of Cancermagister hemocyanin subunits

Abstract Hemocyanin from Cancer magister is composed of two different subunits with molecular weights of 76,000 and 84,000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. These two subunits may play a functional role in the binding of oxygen by hemocyanin.

Hemoprotein quantitation in isolated hepatocytes.

Methods for quantitation of catalase, cytochromes P-450 and b5 and mitochondrial cytochromes a + a3, b561 + b566, and c + c1 in isolated hepatocytes were developed in analogy to methods established for subcellular systems and were used to measure changes in specific hemoprotein concentrations due to pretreatment and to change in incubation conditions. Pretreatment of rats with phenobarbital or 3-methylcholanthrene resulted in increased concentrations of cytochromes P-450 and b5 on a cellular basis, but had no effect on the other hemoproteins. Chronic ethanol pretreatment resulted in increased cytochrome P-450 and decreased cytochromes a + a3 concentrations. Hemoprotein concentrations in hepatocytes decreased following 4-10-h incubations in rotating round-bottom flasks. Rates of decrease were dependent upon both incubation conditions and prior in vivo treatments with phenobarbital or 3-methylcholanthrene.

On the binding of copper by hemocyanin

Abstract One new sulfhydryl group can be determined by amperometric silver titration in several denaturing media, for each four atoms of copper removed from hemocyanin by anaerobic dialysis with neutral cyanide.

Detection of copper on polyacrylamide gels.

Abstract A color test for the localization of copper on polyacrylamide gels is described. The test is based upon the quenching of fluorescence of bathocuproine sulfonate by Cu 1+ and is sensitive to 0.1 nmol of free or protein-bound copper. There is no false positive reaction with 10 nmol of hemeprotein, free Fe 3+ , Fe 2+ , Co 2+ , or Mn 2+ .

The redox potential of liver cytochrome P-450

Abstract The redox potential of phenobarbital-induced rabbit liver cytochrome P-450 has been measured, within microsomes, using dyestuff reduction under anaerobic conditions, and optical and ESR spectrophotometric techniques for determining the ratios of oxidants and reductants in the system. An apparent midpoint potential of about −0.41 volt at pH 7.0 was found. The uncertainty concerning the exact E′o resulted from inability to achieve titration potentials by the dye-stuff technique, below −0.44 volt.

Dopamine-β-hydroxylase: Evidence for binuclear copper sites

Abstract Copper has been progressively removed from dopamine-β-hydroxylase by incubation with chelex resin. The relationship between the activity (A) and the copper/protein ratio (r) has been shown to be of the form A ⊥ r 2 which implies that the enzyme catalysed reaction is second order with respect to protein bound copper. These results support a model for the catalytic mechanism involving binuclear copper sites rather than the four copper atoms in the enzyme acting independently.

Absorption and circular dichroism spectra of different forms of mushroom tyrosinase.

The circular dichroism spectrum of resting mushroom tyrosinase between 800 and 400 nm showed two bands at 755, and 653 nm. The CD spectrum of resting tyrosinase between 400 and 250 nm showed oxygen-sensitive changes at 350 nm upon treatment of tyrosinase with hydroxylamine or hydrogen peroxide. These were similar to changes observed on regeneration of aged hemocyanin by similar procedures. A structural relationship between the active sites of hydroxylamine- or hydrogen peroxide-treated tyrosinase and hemocyanin is suggested by these observations, confirming inferences based upon other studies (Jolly, Jr., R.L., Evans, L.H., Makino, N. and Mason, H.S. (1974) J. Biol. Chem. 249, 335-345 and Schoot Uiterkamp, A.J.M. and Mason, H.S. (1973) Proc. Natl. Acad, Sci. U.S. 70, 993-996).