The action of collagenase on native collagen

Thesis (Ph.D.)--Boston UniversityThe purpose of the research described in this thesis was to determine if the carbohydrate present in collagen was attacked by collagenase, thus indicating whether collagenase was a proteinase or a specific carbohydrase. This in turn would indicate if the carbohydrate of collagen was responsible for the stability or insolubility of collagen. Collagenase was obtained from culture filtrates of Clostridium histolyticum. A procedure was developed for the large scale production of crude collagenase. Ten liters of culture filtrate yielded 3.0-3.5 grams. The collagenase was assayed by gross observation of the degree of dissolution of collagen or by determining the per cent of nitrogen solubilized. The collagenase was active over a pH-range of 6.5-8.5 and was stable at pH 5.5-9.5. In thermal studies collagenase was stable at 45°C for ten minutes. Inhibition studies showed that periodate, sodium citrate and sodium oxalate inhibited the action of collagenase. None of the various monosaccharides tested had any inhibitory effect upon collagenase. Unlike the inhibitory effect of salt on peptic activity, 1 M sodium chloride had no effect upon collagenase activity. Various collagens (rat tendon, horse tendon, bovine cornea) and elastin were isolated and purified by extensive extractions with 0.2 M phosphate buffer (pH 7.4) at room temperature. The composition of hexosamine and hexuronic acid were each determined by two different methods and compared. There was no hexuronic acid present as determined by the carbazole method and only 0.08-0.12 per cent hexosamine. Thus it is unlikely that collagenase or elastase are mucolytic enzymes. Our method of purification of collagen did not decrease its stability or insolubility. Collagen solubilized by acid, base, pepsin or collagen was assayed for free hexosamine, hexuronic acid and hydroxy-proline in relation to the nitrogen solubilized. Neither, acid, base, pepsin or collagenase digestion of purified collagen showed a release of free hexuronic acid or hexosamine. The small amount of hexosamine present was in a bound form and in a 1:1 ratio with hexuronic acid which was also in a bound form. In order to determine if the chemical reactivity of the collagens was different, the three collagens were subjected to different concentrations of acid and base and the dissolution measured. Rat tail collagen was found to be more readily dissolved by acid or base than bovine or horse collagen. Differences in degree of dissolution were not related to differences in carbohydrate content or inhistological organization. The effect of base on collagen was not inhibited by 1 M sodium chloride aswas the effect of acid. In this respect proteolysis by collagenase resembles alkaline degradation of collagen. The data indicate that hexosamine and hexuronic acid are not part of the collagen's structure and are not responsible for its stability or insolubility. Thus the muco-polysacharides of the ground substance do not appear to be necessary for collagen at the intramolecular level or intermolecular level, for the binding of collagen molecules into fibrils