Nitrosation and analysis of amino acid derivatives by isocratic HPLC

Abstract

The objective of this study was to characterize the nitrosation of the classical amino acids by N 2 O 3 . Nitrosation of amino acids results in the formation of mainly a-hydroxy-acids that are suitable for isocratic HPLC analysis and subsequent quantification of amino acids in biological samples. The method is particularly suitable for detection of amino acids in e.g. fermentation media as the a-hydroxy-acids can be quantified in parallel to a variety of other organic substrates and products. The amino acids were transformed into their corresponding a-hydroxy-acids in acidic KNO 2 solutions. The reactions were terminated by NaOH addition and the a-hydroxy-acids separated by isocratic HPLC and quantified by refractive index or UV absorption detection. Nitrosation of 18 of the classical amino acids; glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-methionine, L-serine, L-threonine, L-asparagine, L-glutamine, L-aspartic acid, L-glutamic acid, L-proline, L-cysteine, L-phenylalanine, L-lysine, L-tyrosine, and L-tryptophane formed detectable nitrosation products. L-Lysine, however, needed incubation in 96 mM formic acid to produce a detectable product, while L-phenylalanine had to be incubated in 120 mM HNO 3 and 100 mM HCl. Optimal reaction conditions for most amino acids included 40 min of incubation of up to 5 g L À1 amino acid in 160 mM KNO 2 in 100 mM HCl at 45 C to maximize product yields.

Metabolic engineering of Bacillus subtilis forl‐valine overproduction

“…l ‐Valine was first converted to its α‐hydroxy acid, that is, 2‐hydroxy‐3‐methylbutyric acid, via nitrosation before quantification via high‐performance liquid chromatography (HPLC) as previously described (Ulusoy, Ulusoy, Pleissner, & Eriksen, ) with minor modifications. Culture samples were centrifuged at 13,000 rpm for 10 min, and the cell‐free supernatant was diluted as required.…”

“…L-Valine was first converted to its α-hydroxy acid, that is, 2-hydroxy-3methylbutyric acid, via nitrosation before quantification via highperformance liquid chromatography (HPLC) as previously described (Ulusoy, Ulusoy, Pleissner, & Eriksen, 2016) with minor modifications.…”

Electrochemical (Bio)Sensing of Maple Syrup Urine Disease Biomarkers Pointing to Early Diagnosis: A Review

“…The on-chip LC methodology was also established for AA detection. Separation methods have been conjoined with varied determination processes, incorporating UV [23,24], FL [21,43,44], MS [45][46][47], and electrochemical [25,26,48] detection. Furthermore, MS has been broadly used and has turned out to be the most accepted determination method in AAs analysis.…”

“…The most common methodologies that are used in the determination of branched-chain amino acids and therefore MSUD, are MS/MS, enzyme activity assays, HPLC, capillary electrophoresis, and genetic testing [19][20][21][22][23][24][25][26][27]. Notwithstanding, all the practices are time exhausting and, in a few incidents, crave a huge sample volume.…”

Redox-Mediated Post-Translational Modifications of Proteolytic Enzymes and Their Role in Protease Functioning

“…as a nitroso group. S-nitrosation, O-nitrosation, and N-nitrosation were observed for the 18 amino acids [26], but only S-nitrosation has received significant attention as a PTM of proteins. Nitration, in contrast to nitrosation, is an irreversible modification.…”