Tyrosine decarboxylase. Spectrophotometric assay and application in determining pyridoxal-5'-phosphate.
We have developed a highly sensitive and rapid spectrophotometric assay for tyrosine decarboxylase that can be applied to determining pyridoxal-5'-phosphate. In the assay, tyramine, a product of tyrosine decarboxylation, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a product soluble in toluene whereas tyrosine does not. We determined the amount of tyramine produced enzymatically by reading the absorbance at 340 nm of a toluene extract of the reaction mixture. This method is capable of detecting as low as 2.9 micrograms/mL of the enzyme. Using this method, we find the Km for tyrosine decarboxylase from Streptococcus faecalis to be 3.55 X 10(-4)M. We have also developed a specific and extremely sensitive method for determining pyridoxal-5'-phosphate, a cofactor of the enzyme, by using this spectrophotometric assay with apotyrosine decarboxylase.
D013293:Enterococcus faecalis / Q000201:enzymology; D007700:Kinetics; D011732:Pyridoxal Phosphate / Q000032:analysis*; D013053:Spectrophotometry / Q000379:methods; D014445:Tyrosine Decarboxylase / Q000032:analysis*
D011732:Pyridoxal Phosphate; D014445:Tyrosine Decarboxylase