Oxidation, enzymic with periodate

Horseradish peroxidase (HRP, EC 1.11.17) was obtained from Huka. The oxidation of carbohydrate moieties of enzyme with periodic acid (0.04 mM in 0.05 mM acetate buffer, pH 5.0) was performed according to Zaborsky and Ogletree (1974). The unreacted periodic acid was removed with 0.025-ethylene glycol. The oxidized enzymes were dialyzed against 50 mM phosphate buffer with pH 6.0 for 18 h. 

Glutaraldehyde cross-linked on silica-alumina with (or without) nickel, silicon carbide, or Kieselguhr Glutaraldehyde cross-linked with albumin Reaction of the periodate-oxidized enzyme with poly(4-aminostyrene)... 

To avoid interference with the active site of the enzyme, D-glucose oxidase from Aspergillus niger has been immobilized by way of its carbohydrate moieties. Thus, treatment of the periodate-oxidized enzyme with a styrene derivative afforded a water-insoluble material retaining full enzymic activity and exhibiting enhanced thermal stability at 60 °C. 

Enzymes that contain carbohydrate, such as HRP or GO, may be oxidized with periodate to create reactive derivatives that subsequently can be used to label antibodies or other targeting molecules at their amine groups. The aldehyde-HRP intermediate may be stored for extended periods in a frozen or lyophilized state without loss of activity (either enzymatic or coupling potential). Avoid, however, storage in a liquid state, since polymerization may occur—resulting in precipitation and loss of activity. 

Two aldehydic nucleotide derivatives have found use as affinity labels. The magnesium salt of (64), formed by oxidation of ATP with periodate, is a competitive inhibitor of pyruvate carboxylase with respect to [Mg. ATP2-],100 and (65), obtained from the / -anomer of 5-formyluridine-5 -triphosphate on treatment with alkali, is a non-competitive and reversible inhibitor of DNA-dependent RNA polymerase from E. coli.101 In each case, addition of borohydride gives stoicheiometric covalent linkage of the nucleotide to the enzyme, with irreversible inactivation. It is thought that condensation with lysine occurs to give a Schiff s base intermediate, which undergoes subsequent reduction. 

Exposure of various invertebrate species to high concentrations of petroleum did not induce mixed function oxidase activity. Enzyme activity was stimulated, however, in a number of fish tissues by petroleum. Different permutations can be addressed as to the significance of basal or induced levels of mixed function oxidases and hydrocarbon toxicity. AHH may have a physiological role in enhancing hydrocarbon clearance but may also increase the mutagenic-carcinogenic potential of hydrocarbons. Both of these concepts have been demonstrated in studies with fish (29,30). Induced AHH levels may permit a more rapid oxidative transformation with concomitant "disappearance" of parent hydrocarbons, but potentially toxic metabolites could be retained in tissues for longer periods (31). It is likely that at the enzymic level the... 

The reduced uterofeirin-phosphate complex (Uff-Pi) forms immediately and reversibly and becomes oxidized over a period of hours (90). The reversibility of its formation would be consistent with the competitive inhibition, and a rate-determining oxidation to Ufo-Pi would be consistent with the slow loss of enzyme activity. 

Periodic acid oxidation has proved to be a very useful tool in enzymology since a wide variety of biochemicals contain hydroxyl groups on adjacent carbon atoms. For example, periodate-oxidized ATP (also called adenosine 5 -triphosphate 2, 3 -dialdehyde) has often been used as an alternative substrate or an irreversible inhibitor for a wide variety of ATP-utilizing enzymes. This compound, and many others, are now commercially available, even though they are readily synthesized e.g., periodic acid oxidized ADP, AMP, adenosine, P, P -di(adenosine-5 )pentaphosphate, P, P -di(adenosine-5 )tetraphos-phate, GTP, GDP, GMP, guanosine, CTP, CDP, CMP, etc. In the case of the nucleosides, commercial sources also can supply the dialcohol form of the nucleoside i.e., the nucleoside has first been oxidized with periodic acid and then reduced to the dialcohol with borohydride. 

In some cases where enzymes have a poor degree of surface amino groups, the covalent linkage can be established through the glycosidic chains by periodate oxidation of enzyme with subsequent coupling to activated nylon (Lopez, Braun Klein, 1996). 

Covalent immobilization of lipase on nylon fibers has been done, using the enzymes carbohydrate groups as chemical link. Oxidation of the lipases carbohydrates with periodate provides aldehyde groups for the binding to hydrazide activated nylon (Lopez, Braun Klein, 1996). 

A small proportion of O-D-glucosylribitol was produced directly by hydrolysis of the teichoic acid with alkali ( see Fig. 16) this product is identical with that obtained by dephosphorylation of the hydrolysis mixture. The major products of such a hydrolysis with alkali were the isomeric monophosphates (58) and (59), in which R = 0-D-glucopyranosyl, both of which gave the O-D-glucosylribitol on enzymic dephosphorylation. The isomer (58) reduced 3 molar proportions of periodate, and the ribitol residue was oxidized, whereas the isomer (59) reduced 2 molar proportions of periodate, the ribitol residue being resistant to oxidation. Small proportions of the diphosphates (56) and (57) were also produced. Oxidation of the diphosphate (57) with periodate, followed by treatment with alkali to remove the aldehydic residues, gave a ribitol diphosphate. 

Poly(ribitol phosphate) synthetase has been found in particulate fractions from Staphylococcus aureus H, and Lactobacillus plantatrum.lt ll-m The bulk of the activity in Lactobacillus plantarum was in crude, cell-wall preparations, and the enzyme is apparently located in the membrane, although intimate association with the wall itself has been suggested. Unlike the natural teichoic acid, the enzymically synthesized ribitol phosphate polymer was readily extracted with phenol hydrolysis by acid and by alkali gave the expected products, and oxidation with periodate indicated a chain length of 5-9 units, a value which compares well with that of 8 units for the natural polymer in the walls of this organism. 

Horseradish peroxidase is coupled to IgG antibody in a two-step procedure. In the first step monosaccharide residues in the enzyme are oxidized with periodate to produce aldehyde groups. Then, in the second step, the aldehyde groups are allowed to react with amino groups in the IgG antibody. The Schiff bases formed are reduced and the conjugate is purified by gel filtration. 

A role for the carbohydrate moieties in the catalytic process of an enzyme was suggested many years ago.24 However, studies with several different enzymes19,149,150 have shown that enzymic activity is affected only slightly if the carbohydrate residues are oxidized with periodate. Furthermore, removal of carbohydrate residues with gly-cosidases,1,15 followed by measurements of activity, has shown that the resulting, modified enzymes had specific activities that were essentially unchanged. With ribonuclease B, it has also been con-... 

A two-electron oxidation of N-acetyltyrosine ethyl ester with mushroom tyrosinase, or with periodate, afforded the N-acetyIdopa ester 142, together with the (Z)-enamide 145 and the 6-acetoxydopa amide 146 (Fig. 40) (284). It is assumed that 145 originates from dopaquinone 143 via 144 by tautomerization. Michael addition of acetate to quinone 143 is believed to be the origin of 146. The formation of quinone methide 144 from dopa ester 142 by tyrosinase is reminiscent of the formation of iminochromes and quinone methides catalyzed by this enzyme in their formation from a-methyl dopa ester (285), and such reactions may well occur in mammalian systems. 

Structural details of various blood group substances were revealed by partial hydrolysis, chromatography, and results of periodate oxidation, treatment with various enzymes such as ficin (M50), and acetolysis (Mil, M12, M15-M17, M19-M21, Y4, Y5). They are well described in various reviews on carbohydrates (K16, S39) and blood group substances (Kl, M49, M50). 

Nitrous oxide inactivates the enzyme methionine synthetase, and caution is urged in giving nitrous oxide to patients who may be deficient in vitamin B12. Low serum vitamin B12 concentrations have previously been reported in patients with sickle cell disease, but the reason for this is uncertain. Three cases of peripheral neuropathy have been reported in patients with sickle cell disease who received nitrous oxide (12-14). AU three had a history of frequent painful sickle crises, for which they received nitrous oxide for prolonged periods. Serum vitamin B12 concentrations were slightly reduced in two patients and very low in the third. The patients aU presented with difficulty in walking and paresthesia. Peripheral sensorimotor neuropathy was confirmed by nerve conduction studies. The patients all responded well to vitamin B12 injections and avoiding further exposure to nitrous oxide. Caution is therefore recommended when using nitrous oxide in patients with sickle cell disease or who are suspected of vitamin B12 deficiency. Two cases of polyneuropathy have also been reported after the use of nitrous oxide for 80 minutes and 3 hours in patients who were subsequently found to have pernicious anemia. They both responded well to hydroxocobalamin. 

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