The Suitability of a Quantitative Spectrophotometric Assay for Phenylalanine Ammonia-lyase Activity in Barley, Buckwheat, and Pea Seedlings

Abstract

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 phenvlalanine onto a-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 gM a-ketoglutarate or 500 ItM 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 rulgare), 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. Erez (3) has pointed to possible errors in the widely Used spectrophotometric assay of phenylalanine ammonia-lyase based on increased absorbance at 290 nm of cell-free preparations in pH 8.8 borate buffer (9). PAL2 deaminates L-phenylalanine to produce trans-cinnamic acid which absorbs strongly at 290 nm under these conditions. He suggests that under optimal conditions for PAL activity and in the presence of aminotransferases and a-keto acids, phenylalanine may be diverted into phenylpyruvate which will tautomerize and fcrm an enol tautomer-borate complex interfering with the assay. As we have used this spectrophotometric assay for the quantitative determination of PAL in barley (6-8), we attempted to determine the extent to which traces of a-keto acids present in our crude extracts might lead to the formation of complexes interfering in the assay. PAL levels in peas and in buckwheat have been examined by several techniques (1, 2), and these plants were included for comparative purposes.

Differential expression of four Arabidopsis PAL genes; PAL1 and PAL2 have functional specialization in abiotic environmental-triggered flavonoid synthesis

“…The supernatant was passed through a Sephadex G25 column. The eluate was used for PAL assay similar to Saunders and McClure (1974). The PAL assay was performed at 37 1C for 1 h in an assay mixture containing 500 mL enzyme extract, 450 mL 100 mM Tris-HCl (pH 8.8) and 50 mL 100 mM L-phenylalanine.…”

Disruption of Poly(ADP-ribosyl)ation Mechanisms Alters Responses of Arabidopsis to Biotic Stress

“…Treated tissue was ground and passed through a Sephadex G-25 column (GE Healthcare Life Sciences). PAL activity was measured from eluate as L-Phe converted to trans-cinnamic acid per hour (A 290 ; Saunders and McClure, 1974). Blanks were made similarly, except for the addition of HCl to a final concentration of 0.25 M prior to the addition of L-Phe.…”

"The Activity of Pathogenesis Related Proteins in Smut Resistant and Susceptible Sugarcane (GT54-9) Mutants Induced by Gamma Radiation"

“…The mix centrifuged at 10,000rpm at 4°C for 10minutes then 1ml of the supernatant was mixed with 1ml 30mM of phenylalanine and 1ml borate buffer and incubated for one hour at 40°C for reaction termination, 0.2ml of 5NHCl was added then the volume increased up to 4ml using distilled water. The amount of transcinnamic acid formed in the reaction was measured at 290nm and calculated according to Saunders et al, 23 as the change in absorbance of 0.01 is equivalent to the production of 3.09 n moles of cinnamic acid. The specific activity of the enzyme was expressed as moles of cinnamic acid produced per hour/gm of the tissue.…”