Studies On Pink Copper-protein, Cytochrome B5 And Cytochrome B5 Reductasefrom Human Erythrocytes.
Year: 1970
Type: dissertation
Abstract: Pink copper-protein, cytochrome b^, and cytochrome b^ reductase had been detected in erythrocyte hemolysates prior to this investigation.The results described are concerned primarily with the functions and properties of these three proteins and their relationships to similar proteins within the erythrocyte.A role has been proposed for erythrocyte cytochrome b^ and cytochrome b^ reductase in the reduction of methemoglobin.Purified pink copper-protein sedimented as a single symmetrical peak in the ultracentrifuge, but minor impurities were detected by polyacrylamide disc electro phoresis.The apparent molecular weight of pink copper protein was 31,50% as determined by gel exclusion, and 32,300, as calculated from an S2ofW of 2.74 and a D2gfW of 7.69 x 10"? cm2 sec"1.The visible spectrum, with absorbance maxima located at 372, 503, and 760 mu,disappeared upon reduction of the protein with sodium dithionite or cysteine.The presence of copper, all in the cupric form, was established by EPR spectroscopy and by reaction of the protein with 2,2 * -biqulnoline and sodium diethyldithiocarbamate Fluorescence spectra of the oxidized and reduced forms of pink copper-protein, and studies of the reactions of the protein with phenylhydrazine and sodium diethyldithio carbamate indicated the presence of pyridoxal in a complex with the cupric ion.Although spectrally similar to several amine oxidases, the pink copper-protein exhibited no activity with a number of substrates in assays for amine oxidases.The pink copper-protein was distinguished from erythrocuprein by its spectral, electrophoretic, and chromatographic properties, and by its inability to catalyze the dismutation of superoxide anion.Cytochrome b^ was isolated from the soluble fraction of hemolysates of human erythrocytes,and a method was developed for quantitating the cytochrome in small samples of blood.It was shown that the amount of cytochrome in the cell decreases upon maturation or storage and that cytochrome b^ is not a degradation product of S-protein, a similar B-type hemoprotein from erythrocyte stroma.Purified erythrocyte cytochrome b^ sedimented as a single symmetrical peak in the ultracentrifuge, but minor impurities were detected by polyacrylamide disc electrophoresis.The apparent molecular weight of the cytochrome was 18,400, as determined by gel exclusion, and 14,600, as calculated from an 8^^ of 1.40 and a Dgo,w of 8.6?x 10"? cm^ sec"}-.After reduction with sodium dithlonite or with NADH and cytochrome b^ reductase, the visible absorbance spectrum shows maxima at 423, 52?» and 556mn and a shoulder at 560 mu.The prosthetic group of cytochrome was shown to be protoheme IX by spectral and chromatographic characterization of the isolated heme and its porphyrin product.The enzymatic properties of erythrocyte cytochrome b$ reductase were studied with both dichlorophenolindophenol and cytochrome b^ as electron acceptors.The reductase was shown to contain FAD as a prosthetic group and to be relatively specific for NADH, catalyzing the reduction of cytochrome b^ at a rate 50 times faster with NADH than with NAD PH.From its nucleotide and electron acceptor specificity and from the effects of ionic strength, pH and EDTA on its activity» it was concluded that erythrocyte cytochrome b^ reductase is the same enzyme as NADH dehydrogenase I (NADH diaphorase)» the enzyme known to be lacking in most cases of hereditary methemoglobinemia.Of possible great significance is the finding that the reduction of methemoglobin by erythrocyte cytochrome b^ reductase is stimulated about 80-fold by erythrocyte cytochrome b^.These in vitro findings suggest the following scheme for the reduction of methemoglobin in the intact cell: NADH -> cytochrome b--> cytochrome bc-► methemoglobin, reductase • DEDICATION To my wife, Joyce,whose patience, understanding, encouragement, and support have been of great importance to me throughout my predoctoral training.To my mother,whose sacrifices
DOI: 10.7302/16664
Author: Passon, Philip George
Language: English
Subfield: Biochemistry
Domain: Life Sciences
Open Access status: green
Funder: U.S. Public Health Service
OpenAlex ID: W4387800565