Peroxidase, testing

Experience leads one to agree with Joslyn s conclusion that this so-called regeneration does not contribute to quality deterioration. The writer has repeatedly observed with frozen pea samples, out in the trade for a year and a half and accepted at the time of packing as passing the peroxidase test and still of normal quality, positive reaction times well within the 3.5-minute time limit very frequently the reaction time has been only a matter of seconds. If it should be proved that these observations are the result of enzyme regeneration, this is additional evidence that peroxidase is not responsible for quality deterioration in underblanched products. 

As mentioned above, the name haptoglobin was coined to designate plasma components forming stable Hb complexes in which the Hb has acquired the properties of peroxidase. To describe an unidentified substance simply by the properties of its complexes with only one other substance is unsatisfactory. Hp as well as Hb from different species will presumably vary to some extent in composition, in the stability of their complexes, and in their peroxidase properties. Thus dog Hp, for example, links human or dog Hb, but the latter Hb complex shows only weak peroxidase activity in Jayle s conventional peroxidase test (N5). 

Peroxidase transformation of mono- and dichlorinated anilines have been extensively studied, but there is scarce information about highly halogenated anilines. From several peroxidases tested, only chloroperoxidase from C.fumago was able to transform highly chlorinated anilines [69]. This first report on peroxidase transformation of pentachloroaniline showed that the main product is a polymeric material, and pentachlorophenol and tetrachloro-l,4-benzoquinone are also produced (Fig. 8.3). The mechanism of pentachlorophenol production from pentachloroaniline is still unknown. However, the tetrachloro-l,4-benzoquinone seems to be a product of the pentachlorophenol intermediate and not produced directly from the pentachloroaniline as found in pentachlorophenol peroxidase transformation [69]. The identified products from the chloroperoxidase-mediated transformation of tetrachloroaniline are the polymer, which represented 87-95% of the total mass, pentachloroaniline, and three different dimers, which have been identified as minor products [69]. 

The cytochrome-c peroxidase test offers the advantages of stability of the peroxidase-hydrogen peroxide complex and high specificity for its hydrogen donor, cytochrome c. 

A systematic survey of donor substances with respect to their specificity for peroxidases has not been attempted but, in general, it would seem that all substances that can act as donors for the peroxidase-hydrogen peroxide system are also oxidized by molecular oxygen in the presence of oxidases. Until donors are found that are oxidized exclusively by the peroxidases the presence of oxidases will interfere with all peroxidase tests which are based on measuring the rate of donor oxidation. The assay of peroxidase in the presence of oxidases will be discussed later. 

The oxidation of benzidine to benzidine blue has been used for peroxidase tests by Madelung (234), Begemann (58), Zinn el al. (382), and Farrell (145). But this reaction can hardly be regarded specific and reliable since numerous compounds give the blue color, such as Cu salts (245), alkali metal halides (302), benzoyl peroxide (119), and others. A compound closely related to benzidine, 2,7-diaminofluorene, has been suggested as a donor by Schmidt and Hinderer (306) and is said to be even more sensitive than benzidine itself. The structures of the two compounds are ... 

The oxidation of iodide introduced by Bach and Chodat (38) as a peroxidase test, and used by Reed (284) for a quantitative procedure, has been abandoned as too unspecific. Evidence for this can he found in the papers by Batelli and Stem (52), Wolf and Stoecklin (377), and Martinand (241). 

Place about o-i g. (or 0 1 ml.) of the amine in a test-tube, add o-i ml. of glacial aatic acid to dissolve the amine and then add water until the test-tube is half-full Next place about 0 3 g. of the peroxidase preparation in a very small mortar and grind up with about 15 ml. of water. Filter through a small fluted filter-paper into a test-tube. 

Detecting the presence of small, even invisible, amounts of blood is routine. Physical characteristics of dried stains give minimal information, however, as dried blood can take on many hues. Many of the chemical tests for the presence of blood rely on the catalytic peroxidase activity of heme (56,57). Minute quantities of blood catalyze oxidation reactions between colorless materials, eg, phenolphthalein, luco malachite green, luminol, etc, to colored or luminescent ones. The oxidant is typically hydrogen peroxide or sodium perborate (see Automated instrumentation,hematology). 

Other specific discovery assays have been used such as differential inhibition of a vancomycin resistant S. aureus strain and its susceptible parent, and an assay based on antagonism of the antibacterial activity by N,A/-diacetyl-L-Lys-D-Ala-D-Ala [24570-39-6] a tripeptide analogue of the dalbaheptides receptor. AppHcation of this latter test to 1936 cultures (90) led to the isolation of 42 dalbaheptides, six of which, including kibdelin (Table 3), parvodicin (Table 3), and actinoidin A2 (68) were novel. A colorimetric assay based on competition between horseradish peroxidase bound teicoplanin and the... 

Because overblanching may result in undesirable changes in color, flavor, taste, and texture and the loss of nutritive value, it is as important to avoid overblanching as underblanching. The availability of a method for the detection of overblanching is indicated, but so far as the writer is aware, none exists at the present time. In view of the fact that complete peroxidase inactivation is not required for quality protection, a measurement of residual peroxidase activity might provide the basis for such a test. 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Test strip and immunoflltration devices were developed by Ostermaier et al to detect sulfadiazine, sulfamethazine, and sulfamethoxypyridazine in milk. Direct competitive immunoassay was utilized with sulfonamide-horseradish peroxidase as the detector. The LOD for sulfamethazine for both the dipstick and immunoflltration was 10 pg kg for sulfadiazine, the LOD was 12 pg kg for the dipstick and 30 pg kg for immunoflltration. For sulfamethoxypyridazine, the LOD was 10 pgkg for the dipstick and 20 pgkg for immunoflltration. The devices were found to be suitable for on-site use with undiluted milk. 

Levels of a number of metabolites as well as a number of enzymes in body fluids are indicative of disease conditions. Many of the enzymatic reactions mentioned above have been used in solution clinical assays as well as in test strips.446,497-508 512-515 Assays for hydrogen peroxide and the enzyme peroxidase using NADH and a tetrazolium salt have been de-scribed.509,5io Assays of exogenous substances (e.g., drugs or their metabolites) also utilize this chemistry. The determination of alcohol using alcohol dehydrogenase is an example.511 As mentioned above, the assay of enzyme levels can also be achieved using tetrazolium salts.516-520... 

The molecular masses of heme catalases are usually significantly higher as compared with peroxidases. If expressed in Lg-1s-1, rate constants for the Fem-TAML activators when compared with catalase from beef liver, which has a molecular weight 250,000 gmol-1 (Table IV, entry 13) (89), look very impressive, viz. 17 L g 1 s-1 for 11 vs. 22 L g 1 s 1 for the enzyme. Nevertheless, the catalase-like activity of the Fem-TAML activators can be suppressed by the addition of electron donors -it is negligible in the presence of the substrates tested in this work. In Nature, catalases display only minor peroxidase-like activity (79) because electron donors bulkier than H202 cannot access the deeply buried active sites of these massive enzymes (90). The comparatively unprotected Fem-TAML active sites are directly exposed to electron donors such that the overall behavior is determined by the inherent relative reactivity of the substrates. 

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