226 



G. LELLI AND G. ARANGIO-RUIZ 



Table 1 . The damage of collagen fibrils through 

 papain treatment. 



Table 2. Controls in distilled water. 



Papain 

 Temp. 



Time 

 Effects 



Powder 

 24 C 



24 h 



Saturated 

 solution 



24° C 



20' 24 h 



- + + 

 + 



2 °o solution 

 50 'C 



1 h 



+ 



5h 



+ + 



14 h 24 h 



+ + + + 

 + + + + 



Biochemically the proteolytic action of papain, like that 

 of pepsin, leads to the formation of peptones. By means 

 of activators, particularly hydrocyanic acid, its action 

 becomes intensified, leading to the formation of three 

 times the amount of aminoacids such as tyrosine, tryp- 

 tophane, leucine, glycocole, alanine, proline and arginine 

 as in the proteinic decomposition due to trypsin. The 

 action of papain thus stands between that of trypsin and 

 pepsin (1). 



Papain develops its optimal activity at pH 5 (trypsin's 

 optimum pH 9) with a rapid decrease towards the acid 

 side, and its solutions are usually neutral. 



The collagen used in these studies was obtained by 

 lacerating in distilled water fragments of guinea pig skin 

 and tendons of rabbits and calves. The papain was 

 obtained from the Nutritional Biochemicals Corporation, 

 Cleveland, Ohio, and was used without activators. In 

 some cases we used "papaiotin" (E. Merck A. G., Darm- 

 stadt) which is the almost pure ferment extracted from 

 Carica papaya. 



As according to Willstaetter & Grassmann (7) papain 

 has an optimal temperature of 65-70 C we have experi- 

 mented with different temperatures without, however, 

 reaching such degrees as would be able to produce 

 directly changes in the collagen fibrils (3). 



The properly lacerated material was washed several 

 times in double distilled water after treatment with the 

 enzyme and then chromium shadowed before being 

 examined in the electron microscope. 



The tables 1-2 summarize the results obtained. 



It is clear that the most severe lesions right up to 

 the almost complete destruction of the fibrils are 

 produced by the 2 "„ solution of papain after 14-24 

 hours of exposure in a thermostat at 50C. Altera- 

 tions of a proportionately lesser degree are seen 



Temp. 



Time 

 Effects 



24°C 



50°C 



20' 24 h 



1 h 5 h 14 h 24 h 



in the other groups with lower temperatures and 

 shorter times. 



These electron microscopic results are in complete 

 agreement with what is already known chemically. 



However, we often found even considerable dif- 

 ferences in the severity of the damage to the fibrils, 

 in spite of identical physical and chemical experi- 

 mental conditions, which we could with certainty 

 attribute to differences in the quality of the collagen 

 used. 



The changes we observed ranged from a sort of 

 loss of tone of the fibril, which became flat and as- 

 sumed a tape-like shape (fig. 1 ), to a swelling of varying 

 degree up to a more or less complete dissolution into 

 filaments (fig. 2). In some cases it was chiefly the 

 periphery of the fibrils that was damaged while 

 the central zone tended to stay compact. The periodic 

 structure sometimes persists in the damaged zone, 

 particularly in the axial part of the fibril; in other 

 cases it is completely lost (fig. 3). The lesions de- 

 scribed are sometimes interrupted along the fibrillar 

 axis by relatively normal areas. 



When powdered papain is applied directly to the 

 collagen there is much less damage. It is characterized 

 by the tape-like appearance of the fibrils or by super- 

 ficial erosions without any appreciable swelling. 



These changes in the collagen fibrils are very simi- 

 lar to those produced by other factors, particularly 

 acid or alcaline solutions and moist heat. These too 

 show varying degrees of severity and extension in the 

 same specimen of collagen; they often have a segmen- 

 tal distribution along the axis of the fibrils and range 

 from a simple loss of tone of the fibril to swelling 



Figs. 1-3. Morphological changes in collagen fibrils produced by papain digestion. Fig. 1. Flat, tape-like fibrils. Figs. 2-3. 

 Dissolution of collagen fibrils into filaments, in fig. 3 with loss of the periodic structure. 



