New enzymatic assay of iron in serum.

A new enzymatic method for assaying iron in serum samples, suitable for automated analyzers, is reported. Three reagent mixtures are used: dilution buffer (pH 3.0; ascorbate), reagent 1 (pH 6.7; apoaconitase), and reagent 2 (pH 7.7; citrate, magnesium, and isocitrate dehydrogenase). Sera are diluted with dilution buffer. Fe3+ is liberated from transferrin in sera under acidic conditions, and then reduced by ascorbate. Reagent 1 is added to diluted specimens, and apoaconitase is reactivated by Fe2+ at neutral pH. The resulting solutions are mixed with reagent 2, so that holoaconitase hydrolyzes citrate to isocitrate and the isocitrate and NADP+ are converted to 2-oxoglutarate, NADPH, and CO2. Serum iron is determined linearly up to 70 mumol/L, with within-run CVs < or = 2.4% and day-to-day CVs < or = 2.9%. This method (y) gives results correlating with those of a Reference Method (x) proposed by the International Committee for Standardization in Haematology: y = 0.98x + 0.38 mumol/L (n = 72, r = 0.996, Sylx = 0.63 mumol/L). The mean (+/- SD) serum iron concentrations measured by our method were 18.5 +/- 5.4 and 15.2 +/- 6.0 mumol/L for 63 males and 166 females, respectively.

Reconstitution of pyrroloquinoline quinone-dependent D-glucose oxidase respiratory chain of Escherichia coli with cytochrome o oxidase.

D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent primary dehydrogenase linked to the respiratory chain of a wide variety of bacteria. The enzyme exists in the membranes of Escherichia coli, mainly as an apoenzyme which can be activated by the addition of pyrroloquinoline quinone and magnesium. Thus, membrane vesicles of E. coli can oxidize D-glucose to gluconate and generate an electrochemical proton gradient in the presence of pyrroloquinoline quinone. The D-glucose oxidase-respiratory chain was reconstituted into proteoliposomes, which consisted of two proteins purified from E. coli membranes, D-glucose dehydrogenase and cytochrome o oxidase, and E. coli phospholipids containing ubiquinone 8. The electron transfer rate during D-glucose oxidation and the membrane potential generation in the reconstituted proteoliposomes were almost the same as those observed in the membrane vesicles when pyrroloquinoline quinone was added. The results demonstrate that the quinoprotein, D-glucose dehydrogenase, can reduce ubiquinone 8 directly within phospholipid bilayer and that the D-glucose oxidase system of E. coli has a relatively simple respiratory chain consisting of primary dehydrogenase, ubiquinone 8, and a terminal oxidase.

The 9-carboxyl group of pyrroloquinoline quinone, a novel prosthetic group, is essential in the formation of holoenzyme of D-glucose dehydrogenase.

Availability of different analogues of pyrroloquinoline quinone as the prosthetic group for apo-D-glucose dehydrogenase was examined. The 9-carboxyl group of pyrroloquinoline quinone was shown to be essential for the reconstitution of the enzyme activity. Although the carboxyl group may not be involved in catalytic function, it is quite probable to contribute the binding of the prosthetic group to apoenzyme.

Method of enzymatic determination of pyrroloquinoline quinone.

An improved enzymatic method for the determination of pyrroloquinoline quinone, a novel prosthetic group of some important oxidoreductases, has been developed with cytoplasmic membrane of Escherichia coli K-12, in which D-glucose dehydrogenase (EC 1.1.99.17) was completely resolved to apo-enzyme by EDTA treatment. Incubation of the EDTA-treated membrane with exogenous pyrroloquinoline quinone in the presence of magnesium ions gave a quantitative determination of pyrroloquinoline quinone by assaying the restored D-glucose dehydrogenase activity. This novel enzymatic method was confirmed to be highly reproducible up to 10 ng of pyrroloquinoline quinone and could be applied to a routine assay of pyrroloquinoline quinone.