12 PROCEEDINGS OF THE NATIONAL MUSEUM vol. 122 



changes to gelatin with heating and is then subject to general pro- 

 teolytic degradation. Such materials as acids, some chlorine com- 

 pounds, and strong bases may extensively damage organized collagen 

 fibers and connective tissue structures; degradation in trypsin results 

 from treatment with some of them. Collagenase, which digests 

 native collagen, is apparently not produced by the pancreas. An 

 extensive account of enzyme breakdown of connective tissue is given 

 by Mandl (1961). Limited carbohydrate and fat digestion may be 

 expected. 



In solution the enzymes become inactive, and the rate of inactiva- 

 tion of trypsin increases, with increase in pH and temperature, to a 

 rather rapid loss of activity when the trypsin is in moderate to strong 

 alkaline solutions (Tauber, 1950, p. 18). 



The reaction rate of enzymes depends partly upon the nature of 

 the substrate, the temperature, and the inhibitors present in solution. 

 Inhibition is increased with increase in acidity or alkalinity or with 

 concentration of a nonsubstrate substance beyond the optimum. 

 Tauber (1950, p. 18) has hsted a number of inhibitors of trypsin that 

 are important in this method: charcoal, unsaturated fatty acids, 

 tryptic digests of proteins, pancreatic trypsin inhibitor, hydrogen 

 sulphide, alcohol, formaldehyde, thymol, alkaloids, glycerol, fats, 

 sugar, a number of inorganic salts including heavy metals, ionizing 

 radiation (x-rays, ultraviolet hght), and bacteria. 



The optimum temperature for digestion is probably near the body 

 temperature of the animal from which the enzymes were obtained. 

 At both lower and higher temperatures the activity is slowed, and 

 at higher temperatures the enzymes may be denatured. To prevent 

 thermal denaturization of connective tissue, avoid heating specimens. 

 Temperatures of 30°C or below are recommended for fishes; somewhat 

 higher temperatures will probably not harm reptiles, birds, or mam- 

 mals (see Takahashi and Tanaka, 1953; Takahashi and Takei, 1954; 

 Takahashi and Yokoyama, 1954). 



The pH of the active digestion solution dropped in all cases that I 

 observed, the rate depending upon the enzyme activity and the quan- 

 tity of buffer solution used. After most digestion solutions have 

 become virtually inactive, they cannot be restored to any worthwhile 

 degree of effective activity by the addition of either an ali<;aline solu- 

 tion (to elevate pH) or by the addition of more enzjmie. But the 

 relatively inactive enzymes in alcohol solutions may be activated to 

 some extent, even after several months, by shght dilution of the 

 alcohol and by adjustment of the pH toward the optimum. 



A number of buft'ers suitable for enzymatic and histochemical 

 studies are described by Gomori (1955). Tests of a number of alka- 

 line materials, including common chemicals, some of the buffers 



