Pyrophosphate amplification reaction for measuring amino acid concentrations with high sensitivity using aminoacyl-tRNA synthetase from Escherichia coli

ABSTRACT

To measure amino acid concentrations with high sensitivity, the pyrophosphate amplification reaction conditions of histidyl-tRNA synthetase (HisRS) and tyrosyl-tRNA synthetase (TyrRS) were examined. The amount of pyrophosphate produced by reactions involving HisRS and TyrRS was amplified compared with the amount of the initial substrate L-amino acid after the addition of excess adenosine-5′-triphosphate and magnesium ions, with incubation at 50°C in an alkaline pH. The amount of pyrophosphate produced in the HisRS and TyrRS reactions was approximately 24- and 16-fold higher than the initial amount of L-His and L-Tyr, respectively. The pyrophosphate amplification reactions involving HisRS and TyrRS showed high substrate specificity for L-His and L-Tyr, respectively. Products of pyrophosphate amplification were identified as p¹, p⁴-di(adenosine) 5′-tetraphosphate, and adenosine-5′-monophosphate using high-performance liquid chromatography. A strong positive correlation was observed for 0 to 50 μM of L-His and L-Tyr in the pyrophosphate amplification reaction (R = 0.98 and R = 1.00, respectively).

Abbreviations: L-His: L-histidine; L-Tyr: L-tyrosine; aaRSs: aminoacyl-tRNA synthetases; ATP: adenosine-5′-triphosphate; aminoacyl-AMP-aaRS: aminoacyl-adenylate intermediate; Ap₄A, P¹, P⁴-di(adenosine) 5?-tetraphosphate; AMP: adenosine-5′-monophosphate; PAR: pyrophosphate amplification rate