The conformation of the high-sulphur proteins of wool. I. The preparation and properties of a water-soluble metakeratin.

Abstract

Whereas the S‐cyanoethyl derivatives of the high‐sulphur kerateines extracted from Merino wool were virtually insoluble in aqueous buffer solutions, the disulphide form of a high‐sulphur fraction designated Metakeratin‐B(5,0‐8+4) (M‐B(5.0‐8+4)), obtained by dialysing the corresponding kerateine fraction in the presence of oxygen, was soluble in aqueous buffer solutions at pH 8.0. The material was highly aggregated in solution and could be partially disaggregated by 8 M urea. Gel filtration of the meta‐keratin fraction in 8 M urea on Sephadex G‐200 indicated that the molecular weights ranged from values exceeding 100,000 (excluded from column) to about 10,000. The latter value was confirmed by sedimentation measurements. When reduced and alkylated with iodoacetate the S‐carboxymethylkerateine fraction so formed (SCMK‐5(5.o 8.4)) contained chiefly components with molecular weights of about 10,000 as determined by gel filtration. M‐B(5,0‐8.4) showed a minimum solubility at about pH 4.4 in salt solutions, the pH of minimum solubility decreasing to about 3.0 with progressive S‐carboxymethylation of the protein. No evidence could be obtained for the presence in M‐B(5.0.8.4) of asymmetrical disulphide groups of the form CyS‐S‐CH2COOH. The incorporation of 8 M urea in solutions of the metakeratin at pH 8 gave rise to a positive difference spectrum, suggesting that few if any additional tyrosine residues become accessible in this solvent. Optical rotatory dispersion measurements gave a value of–50 for the parameter b0 in the Moffitt‐Yang equation. In the presence of 8 M urea the value of b0 became zero. M‐B(5,0‐8.4) was digested relatively slowly by Pronase P but the initial rate of digestion increased greatly as the disulphide bonds were converted progressively to S‐carboxy‐methyl groups, the greatest change in rate occurring over the range 10 to 20% conversion of the cystine residues to S‐carboxymethylcysteine residues. The wavelength of maximum fluorescence of M‐B(5.0‐8.4) was unaffected by the S‐carboxymethylation but the intensity of the fluorescence was greatly increased by this modification. The ultraviolet absorption curve for the metakeratin showed absorption maxima at about 276 and 330 nm. With progressive S‐carboxymethylation the ab‐sorbance over the whole range of wavelengths decreased, the absorbance peak at about 330 nm disappeared, and an additional peak at about 280 nm appeared. The results suggest that the reduced chains of the high‐sulphur protein fraction from wool, when allowed to oxidize to the disulphide form, tend to fold in a manner resembling native globular proteins. The unit structures so formed appear to be stabilized by intrachain disulphide bonds.