ABSTRACT
Abnormal concentrations of amino acids in blood and urine can be indicative of several diseases, including cancer and diabetes. Therefore, analyses that examine amino acid concentrations are useful for the diagnosis of such diseases. In this study, we developed an enzyme-immobilized, small reactor column for flow analysis of amino acid concentrations. For the recognition of asparagine and lysine, asparaginyl-tRNA synthetase and lysyl-tRNA synthase were immobilized onto microparticles, respectively, and coupled with coloration reagents for spectrophotometric detection. This assay has some advantages in the analytical field, such as the ability to detect small amounts of analyte, allowing for the use of a small reaction volume, and ensuring a rapid and efficient reaction rate. This approach provided selective quantitation of up to 480 µM of asparagine and lysine in 200 mM Tris-HCl buffer (pH 8.0).
Subject(s)
Amino Acids/analysis , Amino Acyl-tRNA Synthetases/metabolism , Bioreactors , Enzymes, Immobilized/metabolismABSTRACT
Analysis of the concentration of free amino acids in biological samples is useful in clinical diagnostics. However, currently available methods are time consuming, potentially delaying diagnosis. Therefore, the development of more rapid analytical tools is needed. In this study, a chemiluminescence detection method for amino acids was developed, and the conditions for the enzyme reaction and assay were examined. For the recognition of each amino acid (here, serine, proline, glycine, asparagine, leucine, and histidine), the corresponding aminoacyl-tRNA synthetase (aaRS) was employed, and multiple enzymatic reactions were combined with a luminol chemiluminescence reaction. This method provided selective quantification from 1 to 20 µM for serine, proline, glycine, and leucine; 1 to 60 µM for asparagine; and 1 to 150 µM for histidine. This assay, which utilized aaRSs for the detection of amino acids, could be useful for simple and rapid analysis of amino acids in clinical diagnostics.
Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Asparagine/analysis , Glycine/analysis , Histidine/analysis , Leucine/analysis , Proline/analysis , Serine/analysis , Biological Assay , Buffers , Humans , Luminescence , Luminescent Measurements , Luminol/chemistry , Sensitivity and Specificity , Solutions , WaterABSTRACT
The analysis of free amino acids in urine and plasma is useful for estimating disease status in clinical diagnoses. Changes in the concentration of free amino acids in foods are also useful markers of freshness, nutrition, and taste. In this study, the specific interaction between aminoacyl-tRNA synthetase (aaRS) and its corresponding amino acid was used to measure amino acid concentrations. Pyrophosphate released by the amino acid-aaRS binding reaction was detected by luminol chemiluminescence; the method provided selective quantitation of 1.0-30 µM histidine and 1.0-60 µM lysine.
Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Histidine/analysis , Luminol/chemistry , Lysine/analysis , Calibration , Diphosphates/chemistry , Enzyme Assays , Hydrogen Peroxide/chemistry , Inorganic Pyrophosphatase/chemistry , Limit of Detection , Luminescence , Luminescent Measurements , Protein Binding , Pyruvate Oxidase/chemistry , Reproducibility of Results , Solutions , TemperatureABSTRACT
Inherited loss-of-function mutations in the Rim15p-mediated stress-response pathway contribute to the high fermentation rate of sake yeast strains. In the present study, we found that disruption of the RIM15 gene in ethanol-producing Saccharomyces cerevisiae strain PE-2 accelerated molasses fermentation through enhanced sucrose utilization following glucose starvation.
