Assay of phenylethanolamine N-methyltransferase activity using high-performance liquid chromatography with ultraviolet absorbance detection.

A simple and rapid method for measuring phenylethanolamine N-methyltransferase (PNMT) activity by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection is described. This assay requires a partially purified PNMT preparation derived from bovine adrenals, with noradrenaline and S-adenosyl-L-methionine (SAM) as co-substrates. After incubation, the reaction is stopped by addition of acid and the reaction mixture is analysed directly by HPLC. The enzymatically formed S-adenosyl-L-homocysteine (SAH) is detected at 258 nm and determined. Under optimum conditions, the stability of SAH allowed automation of the HPLC detection. This assay was validated by the determination of the kinetic properties of PNMT. Km values for noradrenaline and SAM defined in this assay (16 and 5.7 microM, respectively) are consistent with previously published values. This assay is simple enough to be used for large series of measurements of PNMT activity testing new methyl acceptors, potential inhibitors or PNMT activity in adrenal medulla.

Effect of linking allyl and aromatic chains to histidine 170 in horseradish peroxidase.

Histidine residues in horseradish peroxidase (HRP) were modified chemically with diethyl pyrocarbonate, 4,omega-dibromoacetophenone or diallylpyrocarbonate. Histidines were chosen as His-170, the fifth ligand of the heme iron atom, forms part of the active site of this enzyme. Good yields of hemoprotein were obtained in all cases. Analysis by HPLC of peptides obtained after tryptic digestion showed that His-170 of HRP was in fact modified. The specific activity remained satisfactory after chemical modification of the histidine residues, and so the active site of HRP can thus be altered without a dramatic loss of hemoprotein or peroxidase activity. This may open routes to the preparation of novel biocatalysts.

Enzymatic amplification for spectrophotometric and electrochemical assays of NAD+ and NADH.

Five different procedures are presented for the enzymatic assay of the sum of NAD+ and NADH concentrations. They are based on the principle of amplification by cycling. The reactions involve oxidation of the formate ion, ethanol, glucose, or carnitine catalyzed by the corresponding dehydrogenases. The detection reactions are based on the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT)/INT-formazan and ferricyanide/ferrocyanide couples and use a diaphorase. Two of the systems presented--with formate ion and ethanol--were coupled with spectrophotometric detection. The absorbance measurement values were multiplied by 3 in the first case and by 20 in the second, with respect to the values that would have been obtained in the same conditions without the amplification system. The accessible concentration ranges were between 0.05 and 100 microM approximately. Three systems--with formate ion, carnitine, and glucose--used an electrochemical detection based on oxidation of the ferrocyanide ion. The response times were of the order of 10 min and the precision of about 5%. The first brought to light some difficulties concerning the design of such devices. For the second, the proportionality constant had a value of the order of 0.25 microA.microM-1 and an accessible concentration range between 0.2 and 40 microM. The third allowed more precise assays for lower concentration values: between 0.02 and 1.5 microM, with a proportionality constant of 0.49 microA.microM-1. Emphasis was placed on the adaptation possibilities of these systems as a function of the assay requirements.

Application of the reaction of dithioesters with epsilon-amino groups in lysine to the chemical modification of proteins.

The reaction of lysine with dithioesters was applied to horseradish peroxidase donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) using carboxymethyl dithiotridecanoate: three to four lysine residues were modified. The modified enzyme was soluble and active in diethyl ether. Papain (EC 3.4.22.2) was modified with carboxymethyl dithiobenzoate: two lysine residues were modified. The modified enzyme was soluble and active in dimethylsulfoxide. From these results it is concluded that dithioesters are efficient reagents for the modification of peripheral lysine residues of proteins. Aromatic dithioesters, less reactive but more selective, should be recommended for thiol-dependent enzymes such as papain.