P-Phenylenediamine oxidase

There is a variety of analytical methods used for ceruloplasmin determinations the most frequently used is the p-phenylenediamine oxidase method, by virtue of its high precision. The oxidation rate of p-phenylenediamine or a derivative is measured spectrophotometrically or gasometrically, determining ceruloplasmin oxidase activity. Sunderman and Nomoto (132) determined plasma ceruloplasmin by measuring... 

Ceruloplasmin by p-Phenylenediamine Oxidase Activity, Clin, Chem, Winston-Salem, N,C, (1970) 16,903-910. 

These observations underline the need for meticulous standardization of enz3Tnatic assay techniques for determining ceruloplasmin. With proper standardization, the enzymatic methods are reliable. However, one must always be alert to the possibility that factors other than the concentration of ceruloplasmin may influence the p-phenylenediamine oxidase activity. 

Ceruloplasmin is an enzyme exhibiting oxidase activity against several substrates with a pH optimum between 5.4 and 5.9. The best known substrate is p-phenylenediamine or its dimethyl derivative. The oxidase activity is much weaker against other substrates such as hydroquinone, catechol, pyrogallol, DOPA, adrenaline, noradrenaline, serotonin (L4). The physiological substrate of ceruloplasmin, if any, has not yet been found. 

Oxidan DCN/WSG. See Sodium dichloroisocyanurate Oxidan TCA/P, Oxidan TCA/SG. See Trichloroisocyanuric acid Oxidase glucose. See Glucose oxidase Oxidation base 10A. See p-Phenylenediamine dihydrochloride... 

Ceruloplasmin. This is the most well-known and yet the least understood copper protein. It is an a2-globulin and there is conclusive evidence that this 132000 molecular weight glycoprotein has just one polypeptide chain [9]. It has 7% carbohydrate. It contains six atoms of copper per molecule. Copper in ceruloplasmin exists in both cupric and cuprous forms. Partial removal of copper from ceruloplasmin results in the loss of its characteristic blue color and loss of enzymatic activity. Ceruloplasmin has enzymatic oxidase activity toward several substrates at pH 5.4-S.9. The best substrate is p-phenylenediamine or its dimethyl derivative. Ceruloplasmin can oxidize ferrous ion to ferric ion. The ceruloplasmin level in the newborn is less than 10 mg/100 mL serum. It rises to adult levels (30.4 5 mg/lOO mL) by 2-4 months of age and continues to rise to a peak at 2-3 years and this declines to adult levels by 12 years of age. The level of ceruloplasmin is affected by various pathological states. Usually, a pronounced deficiency of this protein in serum is characteristic of both Wilson s and Menkes s diseases although normal levels have also been reported in the case of Wilson s disease. 

In the rabbit brain, the Nadi (a-naphthol, dimethyl-p-phenylenediamine) reaction showed marked activity of cytochrome oxidase in the caudate nucleus, the putamen, the anterior nucleus of the thalamus, the optic tectum, the interpeduncular nucleus. Golfs and Burdach s nuclei, and the inferior ohvary nucleus (Shimizu et al. 1957). A moderate reaction was seen in the neocortex, hippocampus, dentate gyrus, substantia nigra, cerebellar cortex and the nuclei of the cranial nerves. 

Laccase. A polyphenol oxidase has been purified from the sap of the lac tree by Keilin and Mann. Laccase differs from the potato and mushroom enzyme in several respects. With regard to substrate specificity, it oxidizes p-phenylenediamine more rapidly than catechol. p-Phenylene-diamine is not a substrate for the other polyphenol oxidases described. Laccase apparently is inert with p-cresol. It is not inhibited by carbon monoxide. Unlike the other phenol oxidases, this enzyme is not a pale yellow, but is blue, as is ascorbic acid oxidase (see below). This enzyme, however, is not an ascorbic acid oxidase. 

Electroactive polyaniline films were synthesized by the catalysis of biUru-bin oxidase (BOD, a copper-containing oxidoreductase). The polymerization of aniline was carried out on the surface of a sohd matrix such as glass sUde, plastic plate, or platinum electrode to form homogeneous films [33]. The BOD was immobilized on the surface by physical absorption. The optimum pH was around 5.5. Some aniline derivatives such as p-aminophenol and p-phenylenediamine were good substrates for BOD. Structural analysis suggested the BOD synthesized polyanihne possessed partially 1,2-substititued structures. Cyclic voltammetric studies demonstrated that the PANl films were electrochemically reversible in redox properties, but differed from that of chemically or electrochemically synthesized PANl. The difference was attributed to the partial 1,2-substitution. Laccases are known to oxidize phenolic compounds in nature in the presence of oxygen and are capable in polyaniline synthesis in vitro [34-36]. 

In 1885, Ehrlich showed that animals after injection of a mixture of p-phenylenediamine and a-naphthol, developed a blue colour in their tissues, owing to formation of an indophenol pigment. Batelli and Stern, in 1912, found that this property was common to almost all mammalian tissue, and ascribed it to the presence of an enzyme, indophenol oxidase. Keilin, in 1929, discovered the significance of the enzyme when he showed that it was able to re-oxidise cytochrome, and hence forms part of an important oxidation system in the living cell. 

The rate of reduction of cytochrome a was examined by addition of various chemical reductants such as p-phenylenediamine, hydro-quinone, and ascorbic acid. These were then tested as substrates for the cytochrome oxidase, the activity of which was measured manometrically. Onlyp-phenylenediamine reduced cytochrome a, while the other reagents tested, except sodium dithionite, had weak reductive activity. In Fig. 11, it is shown that cytochrome a is oxygenated under aerobic conditions and is reduced under anaerobic conditions by addition of 10 Af/>-phenyl-enediamine and the trace of borohydride, used to decrease the concentration of oxygen in the solution in the cuvette. When a trace of oxidized cytochrome c is added to either system, both forms of cytochrome a are instantaneusly oxidized. As shown in Table IX (see Section III.C), the cytochrome a acquires an oxidase activity in cooperation with cytochrome c when the oxygen consumption is measured manometrically in the presence and absence of cytochrome c. 

At between pH 7 and 8, cytochrome a is rapidly oxidized by potassium ferricyanide and reduced by sodium dithionite, like other normal cytochromes. Reduced cytochrome c and p-phenylenediamine are also good reductants of cytochrome a, while borohydride and substrates of cytochrome oxidase such as hydroquinone and ascorbic acid hardly reduce it in the absence of a small amount of cytochrome c. Figure 10 shows the... 

C. Malitesta, F. Palmisano, L. Torsi, and P. Zambonin, Glucose fast-response amperometric sensor based on glucose oxidase immobilized in an electropolymerized poly(o-phenylenediamine) film. Anal. Chem. 

J.P. Lowry and R.D. O Neill, Partial characterization in vitro of glucose oxidase modified poly(phenylenediamine)-coated electrodes for neurochemical analysis in vivo. Electroanalysis 6, 369— 379 (1994). 

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