Specific Enzymatic Determination of Some Alpha-Amino Acids by an Automatic Spectrophotometric Reaction Rate Method.

Abstract

An automatic spectrophotometric reaction rate method is described for the selective enzymatic determination of about 10-7 equivalents of some L-a-amino acids. The method is based on the coupled enzyme reaction in which oxidative deamination of certain L-a-amino acids is specifically catalyzed by L-amino acid oxidase to form hydrogen peroxide, which reacts with o-dianisidine in the presence of horseradish peroxidase to form a colored reaction product which has its absorption maximum at 440 µ. The time required for the reaction to produce a small fixed amount of colored product, and therefore for the absorbance to change by a preselected amount (about 0.06 unit), is measured automatically and related directly to the initial amino acid concentration. Small volumes (about 4 ml.) of nine different L-a-amino acid solutions were determined in the concentration range of 4 to 50 p.p.m. with relative errors

Enzymatic Decarboxylation of Tyrosine and Phenylalanine To Enhance Volatility for High-Precision Isotopic Analysis

“…At least nine amino acid deaminating enzymes are available commercially, specifically l -alanine dehydrogenase, phenylalanine dehydrogenase, asparaginase, lysine oxidase, leucine dehydrogenase, glutaminase, tryptophanase, histaminase, and aspartate amino transferase. Additionally, l -amino acid oxidase produces the α-oxoacid of many amino acids, specifically, leucine, isoleucine, norleucine, α-aminobutyric acid, phenylalanine, tyrosine, tryptophan, norvaline, methionine, histidine, and citrulline, and may apply also to serine, threonine, aspartic acid, glutamic acid, lysine, and ornithine; many of the resulting oxoamino acids may provide sufficiently volatile for GC analysis. d -Amino acid oxidase applies to several d -amino acids as well.…”

Light Absorption Spectrometry

“…In the presence of acetic anhydride, only the tertiary amine reacts if salicylaldehyde is present, primary amines do not react, thus permitting determination of mixtures by use of three aliquots. Methyl orange has also been used to determine Schiff bases (234) Ethyl alcohol (283), alpha amino acids (284) and glucose (285) have been determined by automatic spectro-photometric reaction rate methods in which enzymes are employed.…”

“…Fortyseven derivatives of 2,2'-biquinoline and copper(I) were examined spectrophotometrically to determine the effect of kind and position of substituents on absorption spectra (819). Seven reagents were studied critically in respect to their suitability in determining small amounts of copper in textiles (285). Eight polyphenolic reagents for the spectrophotometric determination of iron(III) and titanium(IV) were compared as to absorbance maximum and molar absorptivity (443).…”

Purification and properties of nitroalkane-oxidizing enzyme from Hansenula mrakii

“…Hydrogen peroxide was determined by the method of Malmstadt and Djiioannou (11). About 13 nmol of hydrogen peroxide could be detected at the lower limit.…”