Purification and Properties of Fructosyl-amino Acid Oxidase fromCorynebacteriumsp. 2-4-1

Abstract Background: Previous methods to measure glycohemoglobin (GHb) have been time-consuming or imprecise; we therefore developed a new enzymatic assay for GHb. Methods: Blood cells were first hemolyzed, and hemoglobin was digested with protease to yield fructosyl amino acid. Fructosyl amino acid oxidase acts on the fructosyl amino acid and generates hydrogen peroxide, which reacts with chromogens in the presence of peroxidase. Total hemoglobin was measured spectrometrically in the same reaction tube. The results were reported as the ratio of the concentrations of GHb and hemoglobin. Results: The measured values were comparable to those determined with a HPLC method and with an immunoassay in blood samples from 2854 patients with diabetes. Regression analysis for the enzymatic assay (y) vs the HPLC method (x) produced the following: r = 0.979; slope, 0.994 [95% confidence interval (CI), 0.986–1.001]; y-intercept, 0.04% (95% CI, −0.09% to 0.01%); n = 2854. For the enzymatic assay (y) vs the immunoassay (x), the regression statistics were as follows: r = 0.982; slope, 1.002 (95% CI, 0.995–1.009); y-intercept, 0% (95% CI, −0.05% to 0.05%); n = 2854. Conclusions: The values measured by the new enzymatic assay are sufficiently correlated with those of the conventional HPLC method and immunoassay, but the proposed assay for GHb is rapid and has high precision.

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Review of Fructosyl Amino Acid Oxidase Engineering Research: A Glimpse into the Future of Hemoglobin A1c Biosensing

Journal of Diabetes Science and Technology ◽

10.1177/193229680900300324 ◽

2009 ◽

Vol 3 (3) ◽

pp. 585-592 ◽

Cited By ~ 36

Author(s):

Stefano Ferri ◽

Seungsu Kim ◽

Wakako Tsugawa ◽

Koji Sode

Keyword(s):

Amino Acid ◽

Hemoglobin A1c ◽

Point Of Care ◽

Detection Methods ◽

Future Research ◽

Acid Oxidase ◽

Amino Acid Oxidase ◽

Engineering Research ◽

Glycated Proteins ◽

Fructosyl Amino Acid Oxidase

Glycated proteins, particularly glycated hemoglobin A1c, are important markers for assessing the effectiveness of diabetes treatment. Convenient and reproducible assay systems based on the enzyme fructosyl amino acid oxidase (FAOD) have become attractive alternatives to conventional detection methods. We review the available FAOD-based assays for measurement of glycated proteins as well as the recent advances and future direction of FAOD research. Future research is expected to lead to the next generation of convenient, simple, and economical sensors for glycated protein, ideally suited for point-of-care treatment and self-monitoring applications.

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Proteins of the kidney microvillar membrane. Purification and properties of carboxypeptidase P from pig kidneys

Biochemical Journal ◽

10.1042/bj2290251 ◽

1985 ◽

Vol 229 (1) ◽

pp. 251-257 ◽

Cited By ~ 23

Author(s):

S Hedeager-Sørensen ◽

A J Kenny

Keyword(s):

Amino Acid ◽

Horseradish Peroxidase ◽

Immunoaffinity Chromatography ◽

Maximum Activity ◽

Single Step ◽

Acid Oxidase ◽

Amino Acid Oxidase ◽

Purification And Properties ◽

Triton X ◽

Polypeptide Chains

Carboxypeptidase P has been purified by immunoaffinity chromatography from pig kidneys. A single-step assay with Z-Pro-Met (where Z represents benzyloxycarbonyl) as substrate was used, methionine being determined by using L-amino acid oxidase and horseradish peroxidase. The enzyme constitutes about 1.5% of the kidney microvillar proteins. Triton X-100-solubilized and papain-released forms of the enzyme were isolated. The former had an apparent subunit Mr of 135 000, and the latter form contained two polypeptide chains of Mr 128 000 and 95 000. The undenatured forms were dimeric proteins. In common with other microvillar hydrolases, carboxypeptidase P was a glycoprotein and each subunit contained one Zn atom. MnCl2 (1 mM) in the assay was necessary for maximum activity; in its absence, 0.5 mM-ZnSO4 produced a limited activation, but was inhibitory at higher concentrations. The Km for Z-Pro-Met, in the presence of MnCl2, was 4.1 mM, and the kcat. for freshly prepared enzyme was 1230 min-1. The enzyme lost activity during storage at −20 degrees C. In a limited survey of peptides, hydrolysis was observed only with substrates containing a proline, alanine or glycine residue in the P1 position, and these included angiotensins II and III. The best substrate in this series was Val-Ala-Ala-Phe.

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