Possible Errors in Quantitative Determination of Phenylalanine Ammonia-Lyase Activity by Spectrophotometric Methods

Abstract

A possible error in spectrophotometric determination of cinnamate, the product of phenylalanine ammonia-lyase activity, using nonpurified protein extracts has been shown.Under optimal conditions for phenylalanine ammonia-lyase activity, with borate buffer and in the presence of ao-keto acids, phenylpyruvate is produced and its enol tautomer-borate complex formed, strongly absorbs at 290 nanometers.idoxal phosphate to the reaction system (4) was not confirmed for phenylalanine (5) or the related tryptophan (14) transaminases. PLP will interfere in,the estimation of trans-cinnamate at 290 nm only under conditions that will drive the keto-enol equilibrium of PLP towards the enol tautomer. This will happen under acidic conditions, under very basic conditions (12), or in the presence of borate (8). The complex enol-borate formed absorbs strongly at 290 nm with X maximum at 298 nm. The possible interference between the two enzymes has therefore been investigated in crude extracts of peach apices in borate buffer, pH 8.8. MATERIALS AND METHODSPhenylalanine ammonia-lyase, first described in plant tissue by Koukol and Conn (9), has received much attention during the last decade mainly because of its strategic position in the biosynthetic pathway of polyphenols and lignin (10) and because of the strong effect light has on its synthesis (3,17). Physiological interest in this enzyme led to the development of rapid methods for determination of PAL2 activity. The method used by Zucker (17) of determining the absorption of the trans-cinnamate formed at 290 nm in the reaction mixture has been followed in most cases (2,11,15). His reaction mixture consisted of borate buffer pH 8.8, L-phenylalanine, and enzyme extract. The wavelength of 290 nm was chosen in order to avoid the absorption of phenylalanine that interferes with X maximum of cinnamate at 268 nm (17). In later work Zucker replaced the acetone powder preparation by direct aqueous extract because of the rapidity of this procedure, as well as the decrease in variation between samples as compared with the acetone powder method (18,19). In crude aqueous extracts, it may be expected that a-keto acids will be present, thus furthering the activity of PT in the reaction mixture determining PAL. Purifying crude extracts with active carbon (11) did not remove a-keto acids from the enzyme preparation. In these cases PLP and an amino acid corresponding to the keto acid present will be produced. PT is widespread in plants (5, 6, 10). Its pH optimum is 8.0. Furthermore, PT has a rather broad substrate specificity for the keto acid-amino group receptor, and it transaminates phenylalanine with a-KG, pyruvate, and oxaloacetate (4, 5). The Two-year-old plants of the peach cultivar Redhaven, grown in 15-liter containers outdoors, were used. Apices consisting of the meristem and the three youngest leaves were taken for enzymic analyses.Enzymic extract has been prepared essentially according to Wightman and Cohen (16) in phosphate buffer, pH 8.5. The grinding medium included 10 mm...

Phenylalanine Ammonia-Lyase

“…The formation of cinnamic acid from phenylalanine can be followed spectrophotometrically (Koukol and Conn, 1961;Zucker, 1965;Havir and Hanson, 1968a), by gas chromatography (Czichi and Kindl, 1975a), by highpressure liquid chromatography (Murphy and Sutte, 1978), and by thin-layer chromatography when a radioactive substrate is used (Havir et al, 1971). The problems associated with spectrophotometric assays have been discussed (Erez, 1973;Camm and Towers, 1973). The choice of assay method is determined in large part by the levels of activity to be measured and the type of tissue from which the enzyme is extracted.…”

Phenylalanine ammonia lyase as a precursory enzyme of legume stem lignification

“…Bidlack and Buxton (1992) (Kishor 1989;Dalkin et al 1990) and is positive effect on lignification has been established (Grand et al L982;De Jaegher et al 1985;Guerra et al 1985). Recenr work (Morrison and Buxton 1993;Morrison et al 1994 (Buxton and Russell 1988;Bidlack and Buxton 1992 (Erez 1973 (Wagner and Hrazdina 1984). Duplicate protein concentrations, in acetone powder, were determined (Bradford 1976) and multiplied by the acetone powder yield per plant to obtain protein on a per plant basis.…”

Light Control of Anthocyanin Biosynthesis in Zea Seedlings

“…In preliminary experiments, cinnamic acid production was normally linear for up to 4 b. No phenylalanine transaminase activity was found when «-ketoglutarate was added to the reaction mixture, eliminating the possibility of spurious readings due to formation of enol tautomer-borate complexes of phenylpyruvate which absorb strongly at 290 nm as reported by Erez (1973).…”