178 THE BIOCHEMISTRY OF B VITAMINS 



hydrolyzed when heated with dilute acid (0.05iV) . 207 Stronger concentra- 

 tions of acid are less effective. The ester of the amine is hydrolyzed by 

 acid more rapidly than is pyridoxal phosphate. Decomposition of the 

 latter ester was noted when neutral solutions were stored in the refriger- 

 ator. 



H 3 C 



pyridoxal phosphate pyridoxamine phosphate 



Assay Methods. The method generally favored for the enzymatic 

 determination of pyridoxal phosphate is the system in which tyrosine 

 apodecarboxylase is reactivated. The source of apoenzyme is a dried cell 

 powder prepared from Streptococcus faecalis R., which has been grown 

 on an alanine-rich, vitamin B 6 -deficient medium. A detailed description 

 of the assay procedure has been published. 192 The powder is easily pre- 

 pared and is stable over long periods of time. When preparations are 

 assayed with the powder and a substrate, the rate of carbon dioxide 

 evolution is a measure of the amount of pyridoxal phosphate in the 

 preparations. More elaborate procedures are needed to prepare the apo- 

 enzymes of decarboxylases, transaminases, and tryptophanases from 

 normal cells or tissues; this makes their use as testing agents less con- 

 venient than the procedure employing deficient cells. 



A microorganism which responds only to the phosphorylated deriva- 

 tives of either pyridoxal or pyridoxamine has been encountered. 209 The 

 amine phosphate is three to five times as active as the aldehyde ester. 

 The individual determination of each of the unphosphorylated com- 

 ponents of the B 6 group — pyridoxine, pyridoxal, and pyridoxamine — 

 can be accomplished by a differential method in which samples are 

 analyzed by use of three organisms which respond differently to the three 

 compounds. 210 If materials are tested by such a procedure both before 

 and after dilute acid hydrolysis (which cleaves the phosphate ester 

 linkage) , it is possible to get a reasonably accurate estimate of both the 

 pyridoxal phosphate and the pyridoxamine phosphate contents of crude 

 extracts. 207 The ease with which the amine and aldehyde forms can be 

 interconverted, by nonenzymatic as well as enzymatic reactions, should 

 always be considered when interpreting results obtained by this method. 



The formation of the amine phosphate from the aldehyde phosphate 

 has been followed spectrometrically at an alkaline pH. By this procedure 



