A fluorometric assay for HIV-protease activity using high-performance liquid chromatography

A rapid sensitive method for the quantification of in vitro HIV-protease activity has been developed on the basis of the endoproteolytic conversion of N-Dns-SQ-NYPIV to N-Dns-SQNY. The use of the N-dansyl group as a fluorescence label was shown to not significantly alter the apparent kinetic parameters for the peptide-enzyme interaction. Using fluorescence detection, the dansylated product and unconverted substrate are detected in a single rapid (3 min) isocratic reverse-phase HPLC separation in quantities as low as 0.2 pmol. The method is highly reproducible and suited to a variety of applications including the analysis of large sample numbers and rigorous enzymological studies.     

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity.

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses the small synthetic peptide benzyloxycarbonyl-L-glutaminylglycine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. A detection limit of 31 pmol of product per injection was measured. An apparent Km of 34.7 +/- 2.4 mM was determined for the peptide substrate with purified guinea pig liver enzyme. Using this assay, a series of alkyl aldehydes was shown to inhibit transglutaminase. Modification of this assay using either gradient or isocratic elution with various proportions of acetonitrile (0.1% trifluoroacetic acid)/water (0.1% trifluoroacetic acid) afforded assays for a series of glutamine-containing peptides including substance P, alpha-endorphin, and two small, synthetic peptides. The assay is suitable for measurement of transglutaminase activity with purified enzyme or with crude preparations. This method provides a sensitive, quantitative assay for the determination of substrate and inhibitor properties of small peptides toward transglutaminases.     

An assay for mandelate racemase using high-performance liquid chromatography

Mandelate racemase (EC 5.1.2.2) catalyzes the interconversion of the two stereoisomers of mandelic acid. A fixed-time assay for the quantification of mandelate racemase activity has been developed. The assay involves enzymatic conversion of R-mandelate to S-mandelate (or the reverse reaction) followed by separation and detection of the substrate and product using isocratic reversed-phase high-performance liquid chromatography on a Sumichiral OA-6100 column and absorbance detection. This method offers an economical and efficient alternative to the existing circular dichroism-based and coupled assays.     

A highly sensitive high-performance liquid chromatography-- fluorometric method for the assay of peptidylarginine deiminase activity

The activity of peptidylarginine deiminase (PAD) has generally been assayed by a colorimetric method using N-benzoyl-L-arginine ethyl ester (BAEE) and N-benzoyl-L-arginine (Bz-L-Arg) as the substrates. The widespread occurrence of citrulline and urea in tissues makes use of this method difficult, especially for small samples. We developed a highly sensitive high-performance liquid chromatography method with N-dansyl-glycyl-L-arginine as the substrate. This method was sensitive enough to determine previously undetectable activity of PAD in HL-60 cells. Two types of PAD (HL-60 cell and brain PAD) could be distinguished by differential competition, using either BAEE or Bz-L-Arg as a preferential substrate in the assay. These data indicate that the present method is applicable to many tissues.     

Hydroxyindole-O-methyltransferase activity assay using high-performance liquid chromatography with fluorometric detection: determination of melatonin enzymatically formed from N-acetylserotonin and S-adenosyl-l-methionine

A reliable, sensitive and rapid assay has been developed for determining the activity of hydroxyindole-O-methyltransferase (HIOMT; S-adenosyl-l-methionine:N-acetylserotonin-O-methyltransferase; EC 2.1.1.4), which catalyzes the final step in the melatonin (N-acetyl-5-methoxytryptamine) biosynthetic pathway. This method is based on the separation and detection of melatonin formed enzymatically from N-acetylserotonin and S-adenosyl-l-methionine, by high-performance liquid chromatography with fluorometric detection. The detection limit for melatonin formed per sample was as low as 150 fmol, indicating that the sensitivity of this assay was comparable to that of a radioisotopic assay. The assay was applied to the determination of HIOMT activity in rat pineal gland. The HIOMT activity obtained in this study was comparable with, or slightly lower than those reported previously using radioisotopic assays.     

A high-performance liquid chromatography assay for asparagine synthetase

A highly sensitive method for assaying asparagine synthetase and its glutaminase activity is presented. The amino acids L-asparagine, L-aspartate, L-glutamate, and L-glutamine, are separated by derivatization with o-phthaldialdehyde followed by reversed-phase high-performance liquid chromatography on an Altex ultrasphere-ODS C18 column. The elution is isocratic and the mobile phase used is 50 mM sodium acetate buffer (pH 5.9) with 30% methanol. This assay can easily detect picomoles of asparagine, which may be difficult to do with the other assays that have been described.     

A fluorometric determination of N-methyl-4-phenylpyridinium ion, using high-performance liquid chromatography

A simple and sensitive assay procedure for the quantitation of N-methyl-4-phenylpyridinium ion (MPP+), a neurotoxin, was devised using its fluorescence and high-performance liquid chromatography (HPLC). The fluorescence intensity of MPP+ was several thousand times more than that of N-methyl-1,2,3,6-tetrahydropyridine (MPTP), a metabolic precursor of MPP+. This method was found to be sensitive enough to measure less than 10 pmol MPP+ without using HPLC and 10 fmol using HPLC. The oxidation of MPTP by monoamine oxidase in human brain synaptosomal mitochondria was examined by this assay method. This fluorometric-HPLC method should have broad application in the study of the neurotoxin MPP+.     

A reverse-phase high-performance liquid chromatography assay for dihydroxy-acid dehydratase

A sensitive method for assaying dihydroxy-acid dehydratase activity is described. This enzyme produces alpha-ketoisovaleric and alpha-keto-beta-methylvaleric acids, respectively, in the biosynthesis of valine and isoleucine. These alpha-keto acids, after derivatization with 2,4-dinitrophenyl-hydrazine, were separated and quantified by reverse-phase high-performance liquid chromatography on a Zorbax octadecylsilane C-18 column. As little as 50 pmol of alpha-ketoisovaleric was detected in assays using cell-free extracts from Escherichia coli, whose measured specific activity was 8 mumol of alpha-ketoisovaleric acid produced per hour per milligram protein.     

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. d-Tyrosine was used for the control. α-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.     

A colorimetric assay for measuring peptidylglycine alpha-amidating monooxygenase using high-performance liquid chromatography.

In many peptide hormones and neuropeptides, the carboxy-terminal alpha-amide structure is essential in eliciting biological activity. In the present study, a rapid and sensitive assay method for the determination of peptidylglycine alpha-amidating monooxygenase (PAM) activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-NH2 (Dabsyl-Gly-Phe-NH2), enzymatically formed from the substrate 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This method is sensitive enough to measure Dabsyl-Gly-Phe-NH2 at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 5 min per sample for separation and quantitation. The concentrations of copper and ascorbic acid required for maximal enzyme activity were 1 microM and 2 mM, respectively. The pH optimum for PAM activity was 5.0 to 5.5. The Km and Vmax values were respectively 3.5 microM and 100 pmol/micrograms/h with the use of enzyme extract obtained from bovine pituitary. By using this method, PAM activity could be readily detected in a single rat saliva. The sensitivity of this assay method will also aid in the effort to examine the regulation of in vivo PAM activity.     

An assay for dihydroorotase using high-performance liquid chromatography with radioactivity detection

An assay for measuring dihydroorotase activity was devised. Radiolabeled substrate and product were separated by high-performance liquid chromatography using a reverse-phase column with ion-pairing, and the radioactivity was quantitated by flow detection.     

A peptidyl alpha-amidation activity in chromaffin granules of bovine adrenal medulla

Peptidyl alpha-amidation activity in bovine adrenal medulla has been localized in chromaffin granules by density gradient centrifugation. The activity was found to be both soluble and membrane-associated. Both enzymatic activities were stimulated by the addition of Cu2+ and ascorbate. The pH maximum for alpha-amidation in the chromaffin granules in pH 8.0-8.5. By gel filtration, the soluble enzyme activity appeared as a protein of approx. 40 kDa. It is suggested that this enzyme is involved in the carboxyl-terminal amidation of metorphamide, amidorphin and neuropeptide Y.     

An automated assay for adenylate cyclase using reversed-phase high-performance liquid chromatography

An automated high-performance liquid chromatographic method for the assay of 3',5'-cyclic AMP was developed using octylsilica. Total analysis time was 10.1 min, with cAMP eluting at 3 min. As little as 10 pmol of cyclic AMP could be detected by absorption at 260 nm. Peak height and area were linearly related to cyclic AMP concentration over at least two orders of magnitude. The analytical procedure gave good results in the assay of crude microsomal preparations of adenylate cyclase from both bovine brain and sea urchin eggs. The method was used to demonstrate that sea urchin adenylate cyclase is a Ca2+-activated enzyme.     

A sensitive, nonradiometric assay for dihydroorotic acid dehydrogenase using anion-exchange high-performance liquid chromatography

A new method to assay the mitochondrial pyrimidine de novo enzyme, dihydroorotate (DHO) dehydrogenase, which catalyzes the dehydrogenation of DHO, with orotic acid as the product was developed. The assay was optimized using a rat liver mitochondrial preparation. Orotic acid was quantified with high-performance liquid chromatography using an anion-exchange column (Partisil-SAX) with uv detection at 280 nm. Isocratic elution with low phosphate buffer at pH 4.0 was used. The detection limit was 20 pmol per injection, which is comparable to previously described radiometric assays. The HPLC assay was compared with a spectrophotometric assay measuring orotic acid formation in a deproteinized reaction mixture. Absorbance was measured at the optimal wavelength for orotic acid, 278.5 nm. This assay is less sensitive and less specific than the HPLC assay, which can also detect UMP which might be formed from orotic acid in whole homogenates. With both assays kinetic parameters of the enzyme were determined. In the high concentration range (80-1000 microM) both Km and Vmax values were comparable. With the HPLC assay the concentration range was extended down to 12 microM and initial rates could be determined. The apparent Km was about 12 microM. The HPLC assay is also suitable for use in the study of inhibition of DHO dehydrogenase.     

Fluorescent high-performance liquid chromatography assay for lipophilic alcohols

A new ultrasensitive fluorescent derivatization procedure for chromatographic analysis of primary, secondary, and nonpolar tertiary alcohols is described. The procedure uses Bodipy FL in basic dichloromethane solution with Mukaiyama’s reagent (2-chloro-1-methylpyridinium iodide) to form highly fluorescent ester derivatives that can be separated by silica normal-phase high-performance liquid chromatography (HPLC). Rhodamine WT and Oregon green 488 were also useful derivatization reagents. The detection limit for detection of cholesterol and bryostatin by Bodipy FL was less than 1 fmol. The reaction conditions are gentle enough that low concentrations of unstable alcohols such as bryostatin 1 can be measured.     

Measurement of nitric oxide metabolites in brain microdialysates by a sensitive fluorometric high-performance liquid chromatography assay

Nitric oxide (NO), formed from arginine by a specific neuronal NO synthase, is an important neurotransmitter in various regions of the central nervous system. While intracerebral microdialysis is an elegant technique to study local extracellular neurotransmitter concentrations in vivo, NO metabolites (nitrate, nitrite (NO(x))) are difficult to study at high temporal resolution because of low tissue concentrations and small sample volumes. We developed a sensitive fluorometric high-performance liquid chromatography (HPLC)-coupled NO(x) assay adapted for the use in brain microdialysate samples. The assay includes an initial enzymatic step in which nitrate is reduced to nitrite. Nitrite is acidified to N2O3, which reacts with 2,3-diaminonaphthalene to form 1-(H)-naphthotriazole. This reaction product can be readily isolated and quantitated by HPLC with fluorometric detection. The theoretical assay sensitivity is less than 1 nM, but numerous sources of contamination must be eliminated in the sampling and assaying process to reliably monitor brain NO(x) outflow by microdialysis.     

Electrochemical determination of arylsulfatase activity using high-performance liquid chromatography

A highly sensitive assay for arylsulfatase A was developed using high-performance liquid chromatography with electrochemical detection. The retention time of the enzymatic product, p-nitrocatechol, on a reverse phase column was approximately 2.0 min. The assay was able to detect 0.43 pmol p-nitrocatechol in a 20-microliter ethanol extract of the reaction mixture. The coefficients of variation for seven determinations of the intra- and interassays were 9 and 8%, respectively. The levels of arylsulfatase A activity in human saliva samples and leukocyte and platelet lysates determined by this assay were within 6 and 11%, respectively, of the levels determined by a spectrophotometric assay. The high-performance liquid chromatography assay may have utility in measuring arylsulfatase A activity in biological samples with low activity or specific activity or in samples with compounds which interfere with the spectrophotometric assay.     

Assay of nicotinamide deamidase activity using high-performance liquid chromatography

A rapid, simple and reproducible method has been developed for the determination of nicotinamide deamidase activity using high-performance liquid chromatography (HPLC). Nicotinic acid (NA) liberated from nicotinamide (NAA) after a 15-min enzyme reaction was determined directly by HPLC without further separation steps. Both NA, the product, and NAA, the substrate were separated by reversed-phase ion-pair isocratic chromatography and detected at 261 nm. The present method could be applied to the measurement of deamidase activity in crude cell extracts prepared from several bacterial strains. The Michaelis constant of nicotinamide deamidase in Enterobacter agglomerans was 36 μM for NAA. This method is useful for the measurement of nicotinamide deamidase from various sources.