New enzymatic method with tryptophanase for determining potassium in serum.
We established a simple and rapid enzymatic method for measuring potassium ion in serum by using tryptophanase (EC 4.1.99.1) purified from Escherichia coli K12 strain (E. coli K12 IFO 3301). The presence of pyridoxal 5-phosphate promotes this enzymatic reaction, and potassium and (or) ammonium ions further accelerate it, with ammonium and potassium ions providing equivalent acceleration. We eliminated endogenous ammonium ion by using glutamate dehydrogenase (GLDH; EC 1.4.1.4), then produced ammonium ion in the presence of tryptophanase, tryptophan, and pyridoxal 5-phosphate. The concentration of formed ammonium ion, which was proportional to that of potassium ion in sample, was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH; we then read the change of absorbance at 340 nm. The standard curve was linear for potassium ion concentrations up to 7.00 mmol/L. The within-assay variation (CV) was 0.89% at 5.51 mmol/L and 1.32% at 3.37 mmol/L. The day-to-day CVs were 0.99% at 6.85 mmol/L and 1.71% at 3.52 mmol/L. Analytical recoveries ranged from 98.7% to 108.9%. The correlation coefficient between values obtained with this enzymatic assay (y) and by flame photometry (x) was 0.995: y = 0.984x + 0.091 mmol/L (Sy.x = 0.105, n = 100). The presence of hemoglobin, bilirubin, or other cations little affects this system.
Kimura
S
S
Central Laboratory for Clinical Investigation, Osaka University Hospital, Japan.
Asari
S
S
Hayashi
S
S
Yamaguchi
Y
Y
Fushimi
R
R
Amino
N
N
Miyai
K
K
D000641:Ammonia / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D007656:Ketoglutaric Acids / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011188:Potassium / Q000097:blood*; D011732:Pyridoxal Phosphate / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism; D014368:Tryptophanase / Q000378:metabolism
D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D007656:Ketoglutaric Acids; D009249:NADP; D011732:Pyridoxal Phosphate; D000641:Ammonia; D014364:Tryptophan; D005969:Glutamate Dehydrogenase; D014368:Tryptophanase; D011188:Potassium