PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 191 



multiplied by 6.25. This conversion factor is merely an average value 

 based on proteins containing 16 per cent of nitrogen and thus leads to 

 some error. 



Although considerable attention has been given to proteolysis in ani- 

 mals, there are comparatively few studies involving microorganisms. It 

 is believed that native proteins probably do not enter the bacterial cell 

 and that protein utilization reflects the ability of the cell to produce 

 extracellular enzymes capable of hydrolyzing the protein to amino acids, 

 the latter entering the cell during metabolism. Comparatively few bac- 

 terial species produce proteolytic enzymes. The simplest test for proteo- 

 lytic activity is to ascertain the ability of a given bacterial species to 

 liquefy certain proteins such as solidified gelatin, coagulated casein, or 

 coagulated serum. This may be done either by incorporating the protein 

 into the growth medium or by testing the cell-free filtrate of a culture on 

 the protein itself. One may also determine the degree of proteolysis by 

 measuring the amino nitrogen changes either by the Sorensen formol 

 titration (Brown, 1925), which depends on the increase in acidity w^hen 

 neutralized formaldehyde is added to a solution containing ammonia, 

 primary amines, amino acids, or polypeptides, or by the Van Slyke 

 manometric technic (Peters and Van Slyke, 1932), which depends upon 

 the production of gaseous nitrogen when nitrous acid acts on an aliphatic 

 amine. 



Amino acids. The three methods most commonly employed for the 

 quantitative estimation of amino acids are chromatography and ion 

 exchange, microbiological assay, and enzymatic technic. All have their 

 merits and disadvantages. Chromatography and microbiological assay 

 allow estimation of essentially all the amino acids, while the enzymatic 

 methods are limited to a few. 



The technics of chromatography depend upon the differential distribu- 

 tion of amino acids between two phases using some form of supporting 

 column. The paper-partition method is perhaps the simplest and most 

 commonly employed, for it accomplishes both separation and estimation 

 of the various amino acids. A two-dimensional system is usually 

 employed with phenol water and n-butanol-acetic water as the developing 

 solvents. Most amino acid spots are made visible with the ninhydrin 

 spray or dipping technic. The amino acid spots are estimated quanti- 

 tatively by (1) measuring the spot area (Berry et at., 1951) or (2) colori- 

 metric measurement after elution of the spot (Housewright and Thorne, 

 1950). 



Complete discussion of paper chromatographic theory and methods, 

 including solvents, spray reagents, and Rf values, particularly as applied 

 to amino acids, has been pubhshed (Block et aL, 1955; Berry et al., 

 1951; Williams and Synge, 1950). 



