The hydrolysis of collagen by trypsin

Abstract

The generally accepted statement that collagen is not hydrolyzed by trypsin unless previously swollen in acid or alkali, shrunk in hot water, or treated with pepsin, rests entirely on qualitative work by Kühne and Ewald 1 in 1887 and 1890.

Using finely sifted hide powder as a source of collagen, we found that it was readily digested by trypsin in concentrations of the protease exceeding 10 mg. per liter. The hide powder was treated in 10 c.c. conical graduated centrifuge tubes with buffer solutions at various hydrogen ion concentrations, centrifuged, and the volume of powder measured. The buffer was then replaced by a trypsin solution made up in a buffer of known P_H, and digestion carried on, with continuous shaking, at 40.00°. By suitable variation of the buffer we found that the tryptic hydrolysis was not affected by pretreatment of the collagen at different P_H's between 1.1 and 8.9 and that the optimum reaction for the hydrolysis was at P_H 5.9. The timehydrolysis curve for trypsin-collagen is of the same nature as with trypsin and proteins in general; since here the substrate is insoluble though hydrated, tryptic action appears to take place at the surface of the substrate particles, whether these be coarsely or colloidally dispersed. The degree of hydrolysis increases with increasing concentration of trypsin and decreasing size of hide powder particles. Complete hydrolysis was not reached in four periods of 20 minutes each, the experiment being then stopped owing to increasing hydrolysis in the control (hide powder and buffer solution without trypsin).

The shape of the hydrolysis-time curve suggests that the reaction is very slightly reversible. Experiments on pelt, using concentrations of trypsin up to 0.4 per cent., showed measurable hydrolysis of the collagen, although the organized skin structure inhibited diffusion of the enzyme and greatly decreased the speed of the reaction.