The Suitability of a Quantitative Spectrophotometric Assay for Phenylalanine Ammonia-lyase Activity in Barley, Buckwheat, and Pea Seedlings.
It has been suggested by others that the spectrophotometric assay of phenylalanine ammonia-lyase based on changes in absorbance at 290 nm may be complicated by a side reaction involving transamination from phenylalanine onto alpha-keto acids. This would lead to the production of phenylpyruvate which would spontaneously tautomerize and form an enol borate complex absorbing at this wavelength. We find that the inclusion of 1 ml of either 60 mum alpha-ketoglutarate or 500 mum phenylpyruvate in our 3-ml reaction mixtures has no significant effect on the spectrophotometric assay of phenylalanine ammonia-lyase in shoots from young seedlings of barley (Hordeum vulgare), buckwheat (Fagopyrum esculentum), or pea (Pisum sativum). Although these side reactions may be involved in preparations with very low enzyme activity, the spectrophotometric determination of phenylalanine ammonia-lyase based on changes in absorbance at 290 nm appears to be a reliable and sensitive technique in these seedlings.
Saunders
J A
JA
Department of Botany, Miami University, Oxford, Ohio 45056.
McClure
J W
JW