Il6 NITROGEN METABOLISM 



incorporating the material into culture media. The occur- 

 rence of enzymic activity in culture filtrates must not be 

 taken as unequivocal evidence that the enzyme is extra- 

 cellular, particularly if high activity is dependent on pro- 

 longed incubation of the cultures: in such circumstances it 

 is natural to suspect that the organisms may have undergone 

 partial autolysis and thus released intracellular enzymes into 

 the medium. If a culture filtrate contains more than one 

 protease, they may be separated from one another by the 

 usual procedures employed in the purification of enzymes. 

 Very active extracellular proteases are produced by species 

 of Proteus, Clostridium, Bacillus, Pseudomonas and Micro- 

 coccus', less active enzymes are formed by the streptococci 

 and staphylococci whilst the lactobacilli and Enterobacteri- 

 aceae (with the exception of Proteus spp.) apparently pro- 

 duce none. 



Collagenase and gelatinase activity 



The ability of certain bacteria to liquefy collagen and the 

 material derived from it, gelatin, was soon discovered fol- 

 lowing the introduction of gelatin as a means of solidifying 

 culture media [12]. One of the methods employed in the 

 quantitative determination of gelatinase activity makes use 

 of an Ostwald viscometer [13], the method being based on 

 the assumption that changes in the viscosity of a solution of 

 gelatin are an index of proteolytic activity. Such a procedure 

 is indirect, and the observed changes in viscosity may not 

 necessarily be due to the hydrolysis of peptide bonds. It is 

 therefore desirable that viscometric methods should be com- 

 pared with those that are based more directly on the results 

 of proteolytic activity, and in particular on the expected 

 appearance of free amino and carboxyl groups in the system. 

 Suitable methods of this type employ the van Slyke appar- 

 atus for the determination of amino groups and the Sorensen 

 titration procedure for carboxyl groups. Indeed, it is evident 

 from the work of Gorini and Fromageot that changes in 

 viscosity may bear no relation to the appearance of free 

 amino groups (Fig. 8.1), but such a correlation has not often 

 been attempted. 



