Kinetic and stereochemical studies on novel inactivators of C-terminal amidation.

C-terminal amidation, a required post-translational modification for the bioactivation of many neuropeptides, entails sequential enzymic action by peptidylglycine alpha-mono-oxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). Here we introduce novel compounds in which an olefinic functionality is incorporated into peptide analogues as the most potent turnover-dependent inactivators of PAM. Kinetic parameters for PAM inactivation by 4-oxo-5-acetamido-6-phenyl-hex-2-enoic acid and 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid were obtained by using both the conventional dilution assay method and the more complex progress curve method. The results obtained from the progress curve method establish that these compounds exhibit the kinetic characteristics of pure competitive inactivators (i.e. no ESI complex forms during inactivation). On the basis of k(inact)/K(i) values, 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid is almost two orders of magnitude more potent than benzoylacrylate, a chemically analogous olefinic inactivator that lacks the peptide moiety. Stereochemical studies established that PAM inactivation by 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid is stereospecific with respect to the moiety at the P(2) position, which is consistent with previous results with substrates and reversible inhibitors. In contrast, 2, 4-dioxo-5-acetamido-6-phenylhexanoic acid, which is a competitive inhibitor with respect to ascorbate, exhibits a low degree of stereospecificity in binding to the ascorbate sites of both PAM and dopamine-beta-hydroxylase.

Application of cloud-point extraction-reversed-phase high-performance liquid chromatography. A preliminary study of the extraction and quantification of vitamins A and E in human serum and whole blood.

Methods available for quantification of vitamins A and E in serum or blood requires preconcentration and clean-up by liquid-liquid extraction, evaporation of the extract, and reconstitution of the extract in a solvent of choice before analysis. This process not only involves the use of toxic organic solvents but also requires a long sample preparation time. The lipids and other non-polar coextractants often require additional steps for sample clean-up and evaporation, which may cause sample losses. The use of cloud-point extraction eliminates most of these sample clean-up problems. We recently demonstrated that cloud-point extraction (CPE) can be used for extraction and quantification of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins (PCDDs) from human serum. We now demonstrate how CPE can be used with human serum and blood, at volumes as low as 50 microl, and report a methodology for extracting and quantifying two clinically important vitamins, (A and E) from human serum and blood. Vitamins A and E were extracted from human serum and blood by using Genapol X-80 as the cloud-point extractant under salting out conditions. Serum and blood samples were diluted in organic-free water to get sufficiently large sample volumes for CPE. The surfactant-rich phases were separated by centrifugation, and the samples were analyzed by HPLC-UV after deleterious coextractants were removed by precipitating them with acetonitrile. The recoveries of spiked vitamins A and E were found to be 85.6+/-0.4% and 82.6+/-5.2%, respectively. The average concentration of vitamins A and E in a serum pool after correction for recoveries were found to be 43.4+/-1.8 microg/dl (1.5+/-0.1 micromol/l) and 564.3+/-65.3 microg/dl (13.1+/-1.5 micromol/l), respectively. Vitamin A and E concentrations in whole blood were found to be 26.3+/-0.4 microg/dl (0.92+/-0.01 micromol/l) and 457.5+/-15.6 microg/dl (10.6+/-0.4 micromol/l), respectively. These values are comparable with those obtained by the reference method used at the Centers for Disease Control and Prevention. The success of the preliminary study will lead to a comprehensive validation of this method for vitamins A and E in serum and blood.

Pyruvate-extended amino acid derivatives as highly potent inhibitors of carboxyl-terminal peptide amidation.

Carboxyl-terminal amidation, a required post-translational modification for the bioactivation of many neuropeptides, entails sequential enzymatic action by peptidylglycine monooxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). The monooxygenase, PAM, first catalyzes conversion of a glycine-extended pro-peptide to the corresponding alpha-hydroxyglycine derivative, and the lyase, PGL, then catalyzes breakdown of this alpha-hydroxyglycine derivative to the amidated peptide plus glyoxylate. We now introduce the first potent inhibitors for peptidylamidoglycolate lyase. These inhibitors, which can be viewed as pyruvate-extended N-acetyl amino acids, constitute a novel class of compounds. They were designed to resemble likely transient species along the reaction pathway of PGL catalysis. A general synthetic procedure for preparation of pyruvate-extended N-acetyl amino acids or peptides is described. Since these compounds possess the 2,4-dioxo-carboxylate moiety, their solution tautomerization was investigated using both NMR and high performance liquid chromatography analyses. The results establish that freshly prepared solutions of N-Ac-Phe-pyruvate consist predominantly of the enol tautomer, which then slowly tautomerizes to the diketo form when left standing for several days in an aqueous medium; upon acidification, formation of the hydrate tautomer occurs. Kinetic experiments established that these novel compounds are highly potent, pure competitive inhibitors of PGL. Kinetic experiments with the ascorbate-dependent copper monooxygenases, PAM and dopamine-beta-monooxygenase, established that these compounds also bind competitively with respect to ascorbate; however, pyruvate-extended N-acyl-amino acid derivatives possessing hydrophobic side chains are much more potent inhibitors of PGL than of PAM. Selective targeting of N-Ac-Phe-pyruvate so as to inhibit the lyase, but not the monooxygenase, domain was demonstrated with the bifunctional amidating enzyme of Xenopus laevis. The availability of potent inhibitors of PGL should facilitate studies regarding the possible biological role of alpha-hydroxyglycine-extended peptides.

Quantification of polycyclic aromatic hydrocarbons and polychlorinated dibenzo-p-dioxins in human serum by combined micelle-mediated extraction (cloud-point extraction) and HPLC.

Quantification of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins (PCDDs) usually requires preconcentration and cleanup prior to analysis. These procedures often involve using large amounts of toxic organic solvents. The sample preparation from serum is even more complex because of the coextraction of lipids and other nonpolar serum components. We describe the unprecedented use of cloud-point extraction to preconcentrate, extract, and clean up PAHs and PCDDs from human serum using the nonionic surfactant Triton X-100. The samples were analyzed by high-performance liquid chromatography with ultraviolet detection. The phase separation was induced by the addition of salt to the micellar serum solutions. The surfactant-rich phase was treated with acetonitrile and water to precipitate and remove some of the unwanted substances in the serum sample extract without significantly affecting the recoveries of the analytes. The favorable characteristics of cloud-point extraction discussed here strengthen its potential use as an alternative to other techniques of separation.

Oxidation of 9-alkylanthracenes by cytochrome P450 2B1, horseradish peroxidase, and iron tetraphenylporphine/iodosylbenzene systems: anaerobic and aerobic mechanisms.

Variously substituted alkylanthracenes were studied as models for polycyclic hydrocarbon oxidations. 9-Methylanthracene was oxidized to 9-(hydroxymethyl)anthracene, 10-methyl-10-hydroxy-9-anthrone, and anthraquinone in several systems, including (i) NADPH- and O2-fortified rat liver microsomes, (ii) cytochrome P450 (P450) 2B1 Supported by either iodosylbenzene (PhIO) or a mixture of NADPH-P450 reductase, NADPH, and O2, (iii) horseradish peroxidase and either H2O2 or ethyl hydroperoxide, and (iv) a mixture of iron tetraphenylporphine (FeTPP) and PhIO (in anhydrous CH2Cl2/MeOH). The microsomal system also formed dihydrodiols from 9-methyl- and 9-ethylanthracenes. The formation of the three oxidized products by the P450/NADPH/O2 system was dependent upon O2 label from 18O2 was incorporated into the products, and no label from H2(18O) was incorporated. No label from 18O2 was incorporated into the three products in the FeTPP/PhIO system. In the horseradish peroxidase/H2O2 system, the formation of the three products was decreased when O2 was omitted, and label from both H2(18)O and 18O2 was incorporated into all three products. The results are interpreted in terms of three mechanisms. One is used by the FeTPP and P450 systems, with all oxygen transfers coming from an FeO entity. The other two pathways are utilized by horseradish peroxidase and begin with formation of a radical cation, which can undergo reactions either with H2O or with O2 to form the products detected here. The involvement of a 9-methylanthracene radical cation in the P450 and FeTPP pathways is a possibility, but rapid rearrangement and oxygen rebound must be invoked. Comparisons of products from various 9-alkylanthracenes do not provide evidence that one-electron oxidation is an integral part of the epoxidation process with these compounds. The significance of the lack of trapping of radical (by H2(18O) in the P450 reactions to DNA adduct formation is considered.

Interaction of non-conjugated olefinic substrate analogues with dopamine beta-monooxygenase: catalysis and mechanism-based inhibition.

The reaction of dopamine beta-monooxygenase (DBM; EC 1.14.17.1) with the prototypical non-conjugated olefinic substrate, 2-(1-cyclohexenyl)ethylamine (CyHEA) [see Sirimanne and May (1988) J. Am. Chem. Soc. 110, 7560-7561], was characterized. CyHEA undergoes facile DBM-catalysed allylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyHEA-OH) without detectable epoxidation or allylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyHEA-OH) without detectable epoxidation or allylic rearrangement, and with stereochemistry consistent with that of DBM-catalysed benzylic hydroxylation and sulphoxidation. The kcat. of 90 s-1 for CyHEA oxygenation is about 75% of the kcat. for tyramine, the substrate commonly used in assays of DBM activity. DBM-catalysed oxygenation of CyHEA also results in mechanism-based inactivation of DBM, with the inactivation reaction yielding kinact. = 0.3 min-1 at pH 5.0 and 37 degrees C, and a partition ratio of 16,000. Although both CyHEA turnover and inactivation exhibit normal kinetics, CyHEA processing also results in gradual depletion of copper from DBM; however, mechanism-based irreversible DBM inactivation occurs independent of this copper depletion when sufficient copper is present in the assay solution. A likely mechanism for turnover-dependent DBM inactivation by CyHEA involves initial abstraction of an allylic hydrogen to form a resonance-stabilized allylic radical, which can then either partition to product or undergo attack by an active-site residue. Acyclic, non-conjugated olefinic analogues exhibit diminished substrate activity toward DBM. Thus, kcat. for oxygenation of cis-2-hexenylamine, which also produces only allylic alcohol product, is only 14% of that for CyHEA. Similarly, kinact./KI for turnover-dependent inactivation by the acyclic olefin 2-aminomethyl-1-pentene is more than an order of magnitude smaller than that for benzylic olefins. Our results establish that DBM catalyses allylic oxygenation of a number of non-conjugated olefinic substrate analogues with neither epoxidation nor allylic rearrangement occurring. The absence of epoxide products from non-conjugated olefinic substrates implies an inability of the activated copper-oxygen species of DBM to effect radical cation formation from a non-conjugated olefinic moiety. The striking contrast between DBM and cytochrome P-450, which carries out both epoxidation and allylic oxidation with non-conjugated olefinic substrates, is probably a reflection of the differences in redox potential of the activated oxygen species operative for these two enzymes.

Comprehensive two-dimensional gas chromatography for the fast separation and determination of pesticides extracted from human serum.

A comprehensive two-dimensional gas chromatograph with flame ionization detection was constructed and evaluated for the fast separation and analysis of pesticides. A two-stage thermal desorption modulator served as an interface between the two capillary GC columns. By controlling the temperature of the modulator chamber, all sample substances covering a wide polarity and volatility range were modulated without sample breakthrough. Orthogonal separations were achieved with a nonpolar first column and a moderately polar second column. The system allows fast separations of complex mixtures. When we used the method to analyze pesticides extracted from human samples, we achieved complete separation of 15 pesticides in less than 4 min. The flame ionization detector gave detection limits for particular pesticides that ranged from 1.8 to 3.8 pg on-column. The relative standard deviations were from 6.2% to 8.8% over the linear dynamic range.

Field-amplified sample stacking in micellar electrokinetic chromatography for on-column sample concentration of neutral molecules.

On-column concentration of neutral molecules was achieved for the first time in micellar electrokinetic chromatography by means of field-amplified sample stacking. The stacking process was accomplished by dissolving the neutral analytes in a low-concentration micellar solution that was still above the critical micelle concentration. The lower total ionic strength in the sample buffer compared to the electrophoresis buffer allowed the negatively charged micelles to migrate rapidly into the boundary between the sample and the running buffer where they slow down. This field-amplified sample stacking was achieved by using normal or reversed electrode polarity and produced a 75-85-fold increase in sensitivity for 1,2,4,7- and 1,2,4,8-tetrachlorodibenzo-p-dioxins. The peak area counts obtained from the sample stacking process were proportional to the sample volume injected, and the stacking efficiency was dependent on the micellar concentration. The best stacking efficiency was obtained when the micelle concentration was slightly higher than the critical micelle concentration. When the injection volume was relatively small, the normal-polarity stacking procedure produced a higher stacking efficiency. However, when the injection volume was large, reversed polarity produced a higher stacking efficiency because the non-uniform distribution of the electrical field strength had been eliminated.

N-succinimidyl methoxyphenylacetic acid ester, an amine-directed chiral derivatizing reagent suitable for enzymatic scale resolutions.

A chiral derivatizing reagent, N-succinimidyl-2-(S)-methoxy-2-phenylacetic acid ester (SMPA), directed toward reaction with primary amine-containing compounds has been synthesized and characterized. This reagent is suitable for HPLC resolution from enzymatic-scale reactions where only microgram quantities of chiral products may be obtainable. SMPA derivatization was shown to be effective in the resolution of the enantiomers of a number of different racemic compounds. SMPA was used to resolve the diastereoisomeric derivatives of a previously unknown enzymatically oxygenated product, allowing determination of the stereochemical course of the enzymatic reaction. SMPA is easily prepared from an inexpensive, commercially available, and enantiomerically pure precursor with the formation of a shelf-stable crystalline product which is utilizable in water-containing solutions. In addition to its usefulness for micro-determinations, SMPA is useful for preparative-scale resolutions of enantiomers since the reagent is cleaved from the diastereoisomeric derivative by acid hydrolysis.

Interaction of dopamine beta-mono-oxygenase with substituted imidazoles and pyrazoles. Catalysis and inhibition.

The interaction of dopamine beta-mono-oxygenase (DBM) with substrate analogues possessing either imidazole or pyrazole functionalities at the alkyl chain terminus was investigated. 1-(4-Hydroxybenzyl)imidazole (4-HOBI) is an active substrate for DBM, and it exhibits the expected ascorbate- and fumarate-dependencies and normal kinetic behaviour at concentrations up to 10 mM. 4-Hydroxybenzaldehyde was identified as the product formed from 4-HOBI on the basis of h.p.l.c. and g.c.-m.s. analysis, and its formation exhibits the expected 1:1 stoichiometry with O2 consumption. The 4-HOBI/DBM reaction is kinetically comparable with other DBM activities, and 4-HOBI is the first substrate analogue yet reported that exhibits substantial activity though lacking a terminal amino group. Introduction of a methyl substituent at the 2-position of the imidazole ring abolishes substrate activity, probably through a steric effect. 1-(4-Hydroxybenzyl)pyrazole, where imidazole is replaced by the isomeric pyrazole moiety, is a potent DBM inhibitor, and not a substrate. These results represent the first report of an active heterocyclic substrate or inhibitor for this enzyme, and establish the basis for the design of new classes of DBM substrates and inhibitors.

Olefin oxygenation and N-dealkylation by dopamine beta-monooxygenase: catalysis and mechanism-based inhibition.

In an initial communication [May, S. W., Mueller, P. W., Padgette, S. R., Herman, H. H., & Phillips, R. S. (1983) Biochem. Biophys. Res. Commun. 110, 161-168], we reported that 1-phenyl-1-(aminomethyl)ethene hydrochloride (PAME) is an olefinic substrate for dopamine beta-monooxygenase (DBM; EC 1.14.17.1) which inactivates the enzyme in an apparent mechanism-based manner. The present study further characterizes this reaction. The inactivation reaction yields kinact = 0.23 min-1 at pH 5.0 and 37 degrees C and is strictly dependent on reductant (ascorbate) and oxygen. The DBM/PAME substrate reaction (apparent kcat = 14 s-1), shown to be stimulated by fumarate, gives the corresponding epoxide as product, identified by derivatization with 4-(p-nitrobenzyl)pyridine. However, the lack of DBM inhibition by alpha-methylstyrene oxide, and the observation of identical PAME/DBM inactivation rates in the absence and presence of preformed enzymatic PAME epoxide, indicates that free epoxide is not the inactivating species. A structure-activity study revealed that 4-hydroxylation of PAME (to give 4-HOPAME) increases both kinact (0.81 min-1) and apparent kcat (56 s-1) values, while 3-hydroxylation (to give 3-HOPAME) greatly diminishes inactivation activity while retaining substrate activity (apparent kcat = 47 s-1). 4-Hydroxy-alpha-methylstyrene was found to be a DBM inhibitor (kinact = 0.53 min-1) with weak substrate activity (apparent kcat = 0.71 s-1), while 3-hydroxy-alpha-methylstyrene and alpha-(cyanomethyl) styrene were found not to exhibit detectable DBM substrate activity and only weak inhibitory activity. 3-Phenylpropargylamine hydrochloride showed no detectable DBM substrate activity but rapidly inactivated the enzyme. A new substrate activity for DBM was discovered, N-dealkylation of N-phenylethylenediamine and N-methyl-N-phenylethylenediamine, and the lack of O-dealkylation activity with phenyl 2-aminoethyl ether and 4-hydroxyphenyl 2-aminoethyl ether indicates that DBM N-dealkylation proceeds via initial one-electron abstraction from the benzylic nitrogen heteroatom. With this new substrate and inhibitor reactivity information in hand, along with the other known substrate reactions, a DBM oxygenation mechanism analogous to that for cytochrome P-450 is proposed.

Oral hypoglycaemic activity of some medicinal plants of Sri Lanka.

Investigations were carried out to evaluate the oral hypoglycaemic activity of some Sri Lankan medicinal plants. Approximately 40 plants available locally are reputed to have oral hypoglycaemic activity. Of these, the mostly widely used are (a) Salacia reticulata (Celastraceae) (b) Aegle marmelos (Rutaceae) and (c) Momordica charantia (Cucurbitaceae). Aqueous decoctions of these plants were investigated for their ability to lower the fasting blood glucose level and improve the glucose tolerance in laboratory animals. The results indicate that the aqueous decoctions of all three plants possess significant hypoglycaemic effect. The magnitude of this effect showed time related variation with the three plants. The highest oral hypoglycaemic activity and the maximum improvement of the oral glucose tolerance were associated with the extract of Momordica charantia while the least but significant effects were shown by Salacia reticulata.