46 



MACROMOLECULAR COMPLEXES 



ably the splitting off, of one or more peptides of the end regions, 

 perhaps one or both of the hypothetical "end-chains," or portions 

 thereof. Efforts were therefore slanted toward the isolation and 

 characterization of any such peptide products, utilizing chiefly the 

 techniques of paper-curtain electrophoresis and two-dimensional 

 paper chromatography. Preliminary results have been reported re- 

 cently (Hodge et al, 1960). In eyaluating possible methods for 

 detecting and isolating products of protease action, we were guided 

 by several considerations. In the first place, it seems to be generally 

 accepted that the most highly purified collagens still contain a small 



1900 

 u '^00 



1 1500 



a: I 300 



lij 



a. 



I 100 

 (/) 



§ 900 



o 



o 



2 700 



t 500 

 > 



t 300 

 < 



100 



-A- 1 NATIVE COLLAGEN 

 WITH TRYPSIN 



•B-l DEGRADED 

 COLLAGEN WITH 

 TRYPSIN 



-C-i TRYPSIN 

 ALONE 



4 8 12 16 20 24 28 32 36 40 



FRACTION I n nr Ez: 2: 21 3ni 



DISTRIBUTION OF l'" 



Fig. 26. The distribution of P*^^ activity in the effluent from paper-curtain 

 electrophoresis at pH 4.9 in pyridine-acetic acid buffer for (A-1) native calf- 

 skin collagen in solution at pH 7 (tris buffer with 0.5M CaCL.) incubated with 

 trypsin (Worthington, 2X recrystallized) 2 hr at 20° C, followed by iodination; 

 (B-l) skin collagen after thermal denaturation and tryptic digestion following 

 the procedure of Grassmann ef al. (1956), followed by iodination; and (C-1) 

 trypsin alone (Worthington, 2x recrystallized) after incubation at 20° C in the 

 same medium as used for Run A-1, followed by iodination. A control run with 

 native collagen alone gave no significant activity in the region of Fraction I. 

 It can be seen that the peak of activity labeled Fraction I on the acid side of 

 the diagram appears to arise by the action of trypsin on both native and 

 denatured TC. Input at center. (From Hodge ef al., 1960.) 



