Abstract
Abstract The plastocyanin from Phaseolus vulgaris has been shown to consist of a single polypeptide chain of 100 residues to which 1 atom of copper is bound. This plastocyanin is the smallest copper-protein yet reported; its molecular weight is 10,690. The molar extinction coefficient (e597) of the copper chromophore was shown to be 4.5 x 103. The copper content and molecular weight contrast with that of plastocyanin preparations from spinach (mol. wt., 21,000) and Chenopodium album (mol. wt., 11,500), which both contain 2 copper atoms (2, 13). Amino acid analysis indicated that the minimum molecular weight of the polypeptide chain is 10,626. The dansyl-Edman procedure showed that plastocyanin has only the one NH2-terminal sequence, Leu-Glx-Val-Leu, and reduction and carboxymethylation of the apoprotein gave a homogeneous product, as judged by acrylamide gel electrophoresis and Sephadex gel chromatography. The molecular weight of the carboxymethyl protein was similar to the minimum molecular weight value of 10,626. There are 2 residues of methionine per minimum molecular weight, and cyanogen bromide cleavage of the carboxymethyl protein gave 3 fragments, containing 58, 35, and 7 residues. The characterization of these peptides confirmed that the protein contains only one type of polypeptide chain, with a molecular weight of 10,626. Molecular weight values for the native protein of about 11,000 were consistently obtained by Sephadex gel chromatography in sodium acetate buffers at pH 6.0 and of varying ionic strengths. The approach to equilibrium technique yielded a value of 10,790 in sodium acetate buffer, pH 6.0. Variable and high molecular weight values for plastocyanin (up to 16,000) were obtained by Sephadex gel chromatography in sodium phosphate buffers. The reason for these anomalous values is not clear. Frontal analysis studies with Sephadex did not indicate dimerization of the molecule in the phosphate buffer. There was no significant difference in the molecular weight or sedimentation coefficient of plastocyanin in acetate and phosphate buffers as judged by ultracentrifugation.
Highlights
A single band was obtained on polyacrylamide gel electrophoresis in 7.5% and in 15% gels
As tyrosine makes by far the major contribution to the extinction at 278 rnp [45], it is to be expected that pure French bean plastocyanin would have an extinction at 278 mp approximately 1.5 times that of spinach plastocyanin and would have a purity ratio of approximately 1.2
Removal of copper exposes the single sulfydryl group [26]. The reactivity of this group [26] is probably the cause of the heterogeneity of the apoprotein on starch gels [27] and on acrylamide gels (Fig. 2), because the heterogeneity was completely abolished by carboxymethylation (Fig. 2)
Summary
Isolation of PlastocyaninAll purification procedures were carried out at 4’. Plastocyanin was isolated as described previously, with omission of the calcium phosphate gel steps [4]. According to the degree of polyphenolic material [18] contaminating the preparation, the plastocyanin was rechromatographed repeatedly until an &n: A 69’1purity ratio between 1.l and 1.2 was obtained [13]. (b) Sephadex G-75 and G-50 medium, with the use of 0.05 M sodium acetate, pH 6.0. Collodion bags (pore size, 5 ~1 (Membranfilter, Gottingen, West Germany)), under atmospheric pressure, were used initially to concentrate plastocyanin solutions. It was found, that some loss of plastocyanin occurred through the membrane. Dilute plastocyanin solutions were concentrated by dialysis against 0.05 M sodium acetate, pH 6.0; adsorption onto a small column of DEAE-cellulose, 1 x 3 cm; and subsequent elution with 0.05 M sodium acetate-O.5. In all experiments described subsequently, plastocyanin with a purity ratio of 1.l : 1.2 was used
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