COMPARISON OF PEROXIDASES FROM BARLEY KERNEL (Hordeum vulgare L.) AND WHEAT GERM (Triticum aestivum L.): ISOLATION AND PRELIMINARY CHARACTERIZATION

Abstract

Peroxidases (POD) of crude extracts from barley and wheat germ were separated, partially purified using salt fractionation, ion-exchange and hydrophobic interaction chromatographies and their properties examined. Barley and wheat germ POD contained basic, neutral and anionic isoforms as confirmed by isoelectric focusing. Toyopearl-Bug1 650 M chromatography resolved POD into four cationic fractions. Chromatography of wheat germ extract on CM-Sepharose isolated an anionic and a neutral @action. Following chromatography on Concanavalin-A Sepharose, enzymes from both cereals showed diferences in their elution properties. Optimum pH ranges were 4.0 to 5.5 (barley) and 5.3 to > 6.3 (wheat germ) and POD reacted differently under acidic or basic conditions. Their catalytic behavior in the presence of calcium also differed. Kinetics of POD were of Michaelian type with a ping-pong mechanism and Michaelis constants of guaiacol oxidation in the presence of hydrogen peroxide variedfrom one enzymatic group to another.

Purification and Characterization of Cell Suspensions Peroxidase from Cotton (Gossypium hirsutum L.)

“…The pH optima cPOD-I and rPOD-II were therefore thought to be suitable for the environment in which they are localized. Our result was nearly similar to that reported for POD isoenzymes in Korean radish at pH 5.0 and 6.5 [48] and wheat germ at pH 5.3 and 6.3 [65] but slightly higher than POD in potato tuber sprouts at 4.0 [52], red pepper at 4.5 [66], marula fruit at 4.0 [67], and broccoli at pH 4.0 [4]. However, Lopez and Burgos [68] reported that the release of the heme group from the enzyme active site was pH dependent and occurred most rapidly below pH 5.0 and led to loss of POD activity.…”

Polyphenolic Substrates of Cationic and Neutral/Anionic Peroxidases From Barley and Malt Using a Chronometric Method for the Determination of Pod Activity

“…Four different cationic and a neutralJanionic POD fractions were extracted from barley and partially purified as previously described (Billaud et al 1999). Briefly, POD from 10 g of ground barley was extracted with 50 mL 0.1 M phosphate buffer (pH 7.0) and purified by successively using ammonium sulfate treatment, ion-exchange and hydrophobic chromatographies.…”

“…In both assay conditions, resulting activities were compared with responses versus the guaiacolM,O, system for which optimal conditions have been previously reported (Billaud et al 1999). POD activity was given either in pkat i.e., pmoles H202 consumed s-' x d-' enzymatic extract and expressed as the ratio phenolic compoundguaiacol or in % relative to maximum activity.…”

“…Differences in the behavior of barley and malt POD isoforms have already been demonstrated with regard to their kinetic properties for H,O, and phenolic substrate, pH optima for the oxidation of phenolics and heat stability (Clarkson et al 1989Bamforth et al 1991;Billaud et al 1999) as well as changes in the isoenzyme cationic POD pattern within barley tissues during malting have been evidenced (Antrobus et al 1997).…”

Purification, characterization and selected inhibition properties of peroxidase from haricot bean (Phaseolus vulgaris L.)

“…However, there are reports showing that the optimum pH of POD varies; for example, it is pH 5.0 for bitter gourd [35] and pH 7.0 for Raphanus sativus [36] and between pH 5.3-6.3 for wheat germ. [37] Optimum temperature profile and thermal stability For determination of thermal and storage stability, POD activities were measured at different temperatures. As shown in Figure 3C, POD was stable at 10 and 20°C and showed highest activity at 30 o C. After that point, the enzymatic activity was gradually lost at 40°C and no activity was detected at 50 and 60°C ( Figure 3C).…”