Extinction coefficient peroxidase

The enzyme horseradish peroxidase is a hemoprotein and the region of the Soret band exhibits large differences between the position and extinction coefficients of the uncombined and combined forms. Both forms were first studied by spectrophotometry, but the E—S complexes were 0 labile that they could not be examined extensively by any other spectroscopic method. Using rapid-scanning spectrophotometry and rapid mixing, Chance was able to distinguish the spectra of compound I and II and determine the various rate constants of the multistep reaction with rather poor precision. 

A band of this type has been observed for an enzyme-substrate complex ES where the enzyme was represented by the oxidized form of peroxidase cytochrome c, cyt(Fe(III)) and the substrate was the reduced form of cytochrome c, cytj (Fe(II)) [298]. Indeed, on mixing the solution of cyt(Fe(I I)) and cytj (Fe(II)) there appeared a new absorption band with the absorption maximum at Emax = 1.4 eV, the extinction coefficient e = 0.35 M-1 cm-1, and the width a = 0.2 eV. This band was referred [298] to charge transfer via electron tunneling, [cyt(Fe(III))/ cyt, (Fe(II))] -> [cyt(Fe(II))/cytl(Fe(III))]. From a comparison of the data on the intensity of this band with the results of fluorescence measurements, the distance between the iron atoms Fe(III) and Fe (II) in the [cyt(Fe(III))/cyt1(Fe(II))] complex has been estimated to be R 15-20 A and the edge-to-edge tunneling distance Rt = 7 A. 

At pH < 6 or at higher peroxide concentrations, other GSH adducts were also formed but have not yet been identified. Presumably they are formed by further oxidation of GS-CH2-AB and/or GSH adduct formation with other MAB oxidation products. Under optimal conditions in the H202 peroxidase system, 83% of the MAB could be recovered as GS-CH2 AB as calculated from a molar extinction coefficient for GS-CH2 AB of 22,600 M at 395 nm in methanol water 1 1. 

Glutathione-peroxidase activity was measured spectrophoto-metrically at 340nm by an enzyme coupled assay procedure of Paglia and Valentine (28) as modified by Reddy, et al. (26). A molar extinction coefficient of 6.2 x 103cm 1 was used in calculations. 

A modification of the acylneuraminate pyruvate-lyase assay using pyruvate oxidase to generate H2O2, and measurement of this with peroxidase and p-chlorophenol-4-aminoantipyrine has been described (Sugahara et al. 1980). The measurement of the chromophore is at 505 nm and a molar extinction coefficient of 1.14 X 104 was found at this wavelength. This test has been used in conjunction with sialidase for estimation of serum sialic acid (Sugahara et al. 1980), but remains less sensitive than the method coupled with lactate dehydrogenase. 

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