The Sulfhydryl Content of Rabbit and Trout Myosins in Relation to Protein Stability

Abstract

Rabbit and trout myosins, prepared under painstaking conditions to exclude all incidental heavy metal contamination from reagents, water, glassware, and other utensils, contained 42 free sulfhydryl (SH) groups per 5 × 10 5 g when the analysis was carried out with the Ellman reagent, 5,5′-dithiobis-2-nitrobenzoic acid, in the presence of 8 M urea which was 6 mM in ethylenediaminetetraacetic acid (EDTA). In the absence of EDTA, 40 free SH groups were determined, while performic-acid-oxidized myosin preparations yielded 42 cysteic acid (half-cystine) residues by amino acid analysis. The results indicated that no disulfide bonds were present in the native myosin molecules. The specific enzyme activity (ATPase) remained constant at a relatively high value of 1.38 to 1.45 μmoles P 1 per milligram protein per minute over 4 weeks of storage at 0°. Such myosin preparations were also homogeneous in the ultracentrifuge up to 4 weeks.In myosins prepared with two times distilled water and "reagent grade" chemicals which had not been purified by passage over cation exchange resins, the SH content decreased after 1–2 days at 0°, to 37 SH per 5 × 10 5 g of protein, and this oxidized form of myosin showed aggregate peaks in the schlieren optical system of the ultracentrifuge after 3 days of storage at 0°.Trout myosin in the presence of traces of heavy metals was more susceptible to oxidation than rabbit myosin.