Release of reducible crosslinks of collagen by total enzymic hydrolysis

Abstract

Enzymic hydrolysis of cow skin insoluble collagen was done to determine if previously identified reducible Schiff base crosslinks were artifacts of the hydrolytic conditions used. Cow skin insoluble collagen was reduced with NaB 3H 4, and samples were subjected to acidic, alkaline and enzymic hydrolysis. In addition, portions of the enzymic hydrolysate were subjected to a second hydrolysis using acid or alkali. The radioactive elution profiles were studied for changes in the concentrations of known crosslink compounds. The results of these experiments show that enzymic hydrolysis does not appear to release the crosslinkages completely, although all of the major ones are found to be present. Alkaline hydrolysis was done on samples of aldol histidine and histidinohydroxymerodesmosine which had been isolated from acid hydrolysates. Synthetic hydroxylysinonorleucine was hydrolyzed with varying concentrations of HCl and with 5 M NH 4OH for 24, 48 and 72 h. Alkaline hydrolysis results in 60% destruction of the crosslinks, aldol histidine and histidinohydroxymerodesmosine. Synthetic hydroxylysinonorleucine is not affected by acid hydrolysis but it is destroyed in direct proportion to the length of time hydrolyzed in 5 M NH 4OH.