Horseradish peroxidase sequences

Two additional systems were exploited in order to confirm the involvement of free-radical processes during vindoline oxidations. These were the enzyme peroxidase and photochemistry. Horseradish peroxidase (HRP) oxidized both vindoline and 16-O-acetylvindoline in the presence of hydrogen peroxide. Vindoline was converted to the enamine dimer 59 (78). During the reaction, the following sequence of redox reactions occurs ... 

Figure 2. Comparison of P. chrysosporium MnP-1 and other peroxidases at regions near the proximal and distal histidines. The peroxidase sequences used were manganese peroxidase (MnP) (20), cytochrome c peroxidase (CCP) QS), horseradish peroxidase (HRP)...

Mechanisms of catalase and peroxidase catalysis. Attention has been focused on a series of strikingly colored intermediates formed in the presence of substrates. When a slight excess of H202 is added to a solution of horseradish peroxidase, the dark brown enzyme first turns olive green as compound I is formed, and then pale red as it turns into compound II. The latter reacts slowly with substrate AH2 or with another H202 molecule to regenerate the original enzyme. This sequence of reactions is indicated by the colored arrows in Fig. 16-14, steps a-d. 

Quantitative measurement of phospholipids is rare in routine clinical practice but more common in research (e.g., in studies of dietary influences). The choline-containing phospholipids lecithin, lysolecithin, and sphingomyelin, which account for at least 95% of total phospholipids in serum, are readily measured by an enzymatic reaction sequence using phospholipase D, choline-oxidase, and horseradish peroxidase. Kit methods with this enzymatic sequence are available commercially. Before the availability of enzymatic reagents, the common quantitative method involved extraction and acid digestion with analysis of the total lipid-bound phosphorus. ... 

The degradation of cholesterol leads to the production of bile acids which are structurally closely related to various steroid hormones. (3-Hydroxysteroid dehydrogenase (EC 1.1.1.51) catalyzes the NAD+-de-pendent oxidation of 3(3-, 17(3- and some l6(3-hydroxysteroids to the respective ketosteroids. The enzyme has been adsorbed on a carbon electrode modified by NMP+TCNQ and the NADH liberated in the reaction oxidized anodically (Albery et al., 1987a). Campanella et al. (1984) employed an enzyme sequence electrode composed of NAD+-de-pendent steroid dehydrogenase and horseradish peroxidase for assay of 7a-hydroxysteroids. 

Kemp and co-workers (K6) developed a colorimetric detection system that incorporates biotin into one (nested) primer and the sequence for a DNA-binding protein (e.g., the GCN4 gene from Saccharomyces cerevisiae) into the other primer. Amplified DNA is captured on an immobilized affinity reagent and the biotinylated product is detected with avidin-horseradish peroxidase and a chro-mogenic substrate. 

Although the precise mechanism for the enhancement remains unknown, Thorpe and Kricka (T8) have proposed that enhancers render the sequence of events for unenhanced luminol oxidation (see Section 3.1.3) more efficient. This would be consistent with kinetic studies on the reactivity of phenol enhancers with the horseradish peroxidase intermediates Compounds I and II (Hll, VIO). Specifically, the hypothesis is that (a) horseradish peroxidase Compounds I and II... 

In an extension of this work biotin-labeled liposomes were also modified with horseradish peroxidase (HRP) via periodate oxidation chemistry [74]. The HRP loaded biotin-labeled liposomes catalyzed oxidation of 4-chloro-l-naphthol in the presence of H2O2 yielding an insoluble product which precipitated onto and fouled the electrode. FIS was used to monitor resistance of electron transfer to the [Fe(CN 6] redox probe resulting a similar detection limit of 650 fM for a DNA sequence relevant to Tay-Sachs disorder. The authors also extended these various approaches to probe and amplify the signal from single-base mismatches in anal3d e DNA [75]. 

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