Experiments on the methylation and acetylation of wool, silk fibroin, collagen and gelatin

Abstract

Vol. 38 AEROMONAS FERMENTATION 171 butylene glycol, ethanol, acetic and lactic acids, CO2 and H,, with traces of acetoin and succinic acid. The 2:3-butylene glycol is a mixture of the 1-and meso-forms. 2. The products of xylose fermentation are very similar, both quantitatively and qualitatively, to those of glucose fermentation. This is regarded as evidence against a simple C3-C2 split of the pentose molecule. 3. The main products of the fermentation of pyruvic acid are lactic and acetic acids, CO2 and H2. Approximately three molecules of pyruvic acid are oxidized to acetic acid for every one reduced to lactic acid. In a previous paper (Blackburn, Carter & Phillips, 1941) it was shown that wool and silk fibroin could be O-methylated either by contact with methyl bromide or iodide at room temperatures, or with methyl sulphate in buffers within the pH range 2-5-8-5. With both proteins the number of CH3 groups introduced by repeated treatment with methyl sulphate was greater than could be accommodated on the free carboxyl groups of glutamic and aspartic acids, and it was suggested that certain 'activated' peptide linkages methylated as well as the free carboxyl groups of the salt-linkages. CH3I and CH3Br introduced fewer COH groups into wool and silk fibroin than did methyl sulphate. The present paper describes the extension of this work to the methylation of collagen and gelatin by similar methods, and the methylation with methyl sulphate and CH8Br of wool which has bieen chemically modified by alkali, acid, nitrous acid and formaldehyde. In addition, the siaultaneous ace-tylation and methylation of wool, silk fibroin and collagen by the combined action of methanol and acetic anhydride has been investigated. METHODS Methylation of collagen and gelatin with methyl suphate and methyl halides The collagen used in these investigations was a limed hide which had been delimed with acetic acid and then dehydrated in acetone. The gelatin was Coignet's Gold Label. The methylation procedure was similar to that described for wool and silk (Blackburn et al. 1941), with certain modifications to suit the properties of collagen and gelatin. Both proteins were cut into small pieces and acetate buffer was used for the methylations with methyl sulphate. At the end of each period of methylation about 1 g. of collagen was removed, washed in three changes of water and then shaken mechanically with water (25 ml.) for 1 hr. It was then dehydrated with acetone, air-dried and conditioned.* The moisture content of the collagen, the physical properties of which appeared to be unaltered, was determined by Barritt & King's method (1926). During methylation, the gelatin absorbed water and swelled greatly. The resulting gel and solution was dialyzed in cellophan against water for 3 days, the water being changed frequently. The resulting gelatinous mass was dried in vacuo below 60°. Before methylation with either CH3Br or CH3I, the collagen was allowed to reach equilibrium with a borate buffer of pH 8, and then dehydrated with acetone and conditioned. The gelatin was treated dry as bought. After methylation, both proteins were rinsed in several changes of benzene and then conditioned. When proteins are methylated by either CHsBr or CH3I, one halogen anion is liberated for each CH3 group introduced. To provide an additional measure of their degree of methylation, the halogen content of some of the methylated collagens and gelatins was therefore determined by the open Carius method (Peters & Van Slyke, 1932). The methylated gelatin * All proteins described in this paper as conditioned had been exposed to a controlled atmosphere of 210 and 70% R.H. until they attained a constant weight. Although all the analyses were made on the conditioned proteins, the results are recorded as percentages of the anhydrous material.