Bovine Erythrocyte Cupro‐Zinc Protein

Abstract

Bovine erythrocyte cupro-zinc protein was obtained by ethanol-chloroform fractionation of red blood cells and purified by chromatography on fibrous DEAE-cellulose, gel filtration on Sephadex G-75, and rechromatography on microgranular DEAE-cellulose. The protein has a molecular weight of about 33000 by gel filtration, and contains 2 gram atoms of cupric copper and 2 gram atoms of zinc per mole. The amino acid composition of the protein has been determined. The protein has an isoelectric point a t pH 4.95 by isoelectric focussing and is resolved into two "size" isomers, one being a minor component, in polyacrylamide gel electrophoresis. The apoprotein behaves as a molecule of smaller "size" than the native protein components in polyacrylamide gel electrophoresis. The ultraviolet spectra of the native and apoproteins show unusual features. These spectra together with the ultraviolet spectra of partially reconstituted apoprotein indicate a stabilizing effect of the zinc atoms on the structure of the chromophoric system. It is suggested that the copper and zinc atoms are not mutually replaceable at their respective sites in the protein. The near ultraviolet and visible spectra of the native protein have been fitted with Gaussian curves which indicate absorption bands a t 340, 440 and 680 nm. The value of &M/Cu2+ for the 680 nm band has been found to be 120, indicating a cupric complex of high symmetry. man DE 32) were obtained from British Drug Houses Ltd. ; DEAE-Sephadex A-50 and Sephadex G-75 and G-100 from Pharmacia AB; and synthetic ampholyte solution for isoelectric fucossing (Ampholine) from LKB-Produkter AB. Chemicals, where possible, were of analytical grade, and were not purified further except guanidine hydrochloride and iodoacetic acid which were recrystauized from ethanol and n-heptane, respectively. Glass-distilled water was used in large scale preparative work. This water was deionized for analytical work by means of an Elgastat B125 deionizer (Elga Products Ltd.). Preparation of Crude Erythrocyte Cupro-Zinc ProteinFresh ox blood was obtained from a slaughterhouse. Four volumes of blood were collected in a stainless steel bucket containing 1 vol. of 3.8O/, (wlv) sodium citrate. Eight litres of blood were processed at a time. The plasma was separated after centrifuging the blood at 1800 xg for 60 min at 4". The cells were mixed with an equal volume of cold distilled water, and the haemolysate was left overnight a t 4". The haemolysate was then chilled at -20" until ice crystals started to form, and 0.5 vol. of a mixture of ethanol and chloroform (3 : I , v/v), which had been pre-chilled to -2O", was added slowly with continuous stirring. The viscous mixture was diluted with 0.1 vol. of cold 0.9O/, (w/v) NaCl and centrifuged at

Murex trunculus Haemocyanin

“…The bands a t approximately 295 and 305 nm may be assignable to tryptophan residues and there may possible be some connexion with the changes in fluorescence quenching when oxygen reacts with haemocyanin [26,27]. The possibility has been raised of oxygen at the copper site in oxyhaemocyanin interacting with neighbouring tryptophan residues facilitating singlet-triplet conversion in the excited state [27]. However the changes in these circular dichroisrn bands near 300 nm are too much masked by those a t 280 and 346 nm to be of much use in interpreting the oxygenation reaction of haemocyanin.…”

Bovine Erythrocyte Cupro‐Zinc Protein

“…The difference absorption spectrum exhibits a minimum a t about 285nm [1], showing that the state of the tyrosine residues has not altered when the copper and zinc atoms are removed from the protein, although there is a changed contribution at 280 nm, in the circular dichroism spectrum, which may be indicative of a conformational change in the vicinity of this residue. The bands in both absorption and circular dichroism spectra a t 255-265 nm may be due to copper transitions, or to those of cystinyl [31,32] or phenylalanyl [33, 341 residues.…”

Metabolic analysis of the synthesis of high levels of intracellular human SOD in Saccharomyces cerevisiae rhSOD 2060 411 SGA122

“…The molecular weight is 32,500 (Keele et al, 1971). There are two Cu(II) and two Zn(II) atoms per molecule (Bannister et al, 1971). SOD has been shown to exert strong regenerative effects on tissues that have become hardened or fibrotic because of age, disease, or injury (Lefaix, 1993).…”