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 assay for peptidyl alpha-amidation activity using high-performance liquid chromatography

A rapid and sensitive method for the determination of peptidyl alpha-amidation activity has been developed and is based on reverse-phase high-performance liquid chromatographic separation and fluorometric detection. A dansylated tripeptide, N-dansyl-Tyr-Val-Gly-OH, is used as the substrate in the assay and the amount of alpha-amidation activity is determined by quantitating the extent of its conversion to product, N-dansyl-Tyr-Val-NH2. Both product and substrate can be detected in a single assay in quantities as low as 5 fmol by isocratic elution using C-18 reverse-phase columns. The method yields highly reproducible results and requires less than 3 min per sample for separation and quantitation. The assay procedure is applicable to the screening of a large number of samples under different pH conditions and is readily adaptable for use in a variety of studies. For example, the procedure is ideal for detecting alpha-amidation activity in various tissues, monitoring activity at the different stages during purification of a particular alpha-amidation enzyme, determining kinetic parameters of the purified enzyme, and identifying both competitive and noncompetitive inhibitors.     

A fluorescence-based high-performance liquid chromatographic assay to determine acid ceramidase activity.

Acid ceramidase (N-acylsphingosine amidohydrolase) is the lysosomal enzyme required to hydrolyze the N-acyl linkage between the fatty acid and sphingosine moieties in ceramide. A deficiency of acid ceramidase activity results in the lipid storage disorder, Farber disease. This study reports a new assay method to detect acid ceramidase activity in vitro using Bodipy or lissamine rhodamine-conjugated ceramide (C12 ceramide; dodecanoylsphingosine). Using mouse kidney extracts as the source of acid ceramidase activity, this new method was compared with an assay using radioactive C12 ceramide (N-[(14)C]-dodecanoylsphingosine) as a substrate. The Bodipy C12 ceramide substrate provided data very similar to those of the radioactive substrate, but under the experimental conditions tested, it was significantly more sensitive. Using Bodipy C12 ceramide, femtomole quantities of the product, Bodipy dodecanoic acid, could be detected, providing an accurate measure of acid ceramidase activity as low as 0.1 pmol/mg protein/h. Acid ceramidase activities in skin fibroblasts and EBV-transformed lymphoblasts from Farber disease patients were around 7.8 and 10% of those in normal cells, respectively, confirming the specificity of this new assay method. Based on these results, we suggest that this fluorescence-based, high-performance liquid chromatographic technique is a reliable, rapid, and highly sensitive method to determine acid ceramidase activity, and that it could be useful wherever the in vitro detection of acid ceramidase activity is of importance.     

A sensitive fluorometric assay for tissue transglutaminase

We have devised a highly sensitive fluorometric well plate assay for tissue transglutaminase that is suitable for multiple kinetic analyses/high-throughput screening of chemical inventories for inhibitors of this enzyme. The procedure measures the rate of fluorescence enhancement (lambda(exc) 260 nm, lambda(em) 538 nm) when 1-N-(carbobenzoxy-l-glutaminylglycyl)-5-N-(5'N'N'-dimethylaminonaphthalenesulfonyl)diamidopentane (glutaminyl substrate) is cross-linked to dansyl cadaverine (amine substrate). The assay procedure can be used to measure the activity of as little as 60 microU of purified guinea pig liver tissue transglutaminase (4.2 ng or 54 fmol of enzyme).     

A direct fluorometric assay for tissue transglutaminase

Herein we report the design of a direct and continuous fluorometric assay for determining tissue transglutaminase (TGase) activity. The progress of the TGase-catalyzed reaction of 4-(N-carbobenzoxy-l-phenylalanylamino)-butyric acid coumarin-7-yl ester was monitored as an increase of fluorescence (lambda(exc) 330 nm, lambda(em) 460 nm) due to the release of 7-hydroxycoumarin. Using this assay, we determined the K(m) of two acceptor substrates, N-acetyl-L-lysine methyl ester and aminoacetonitrile. We also determined the K(m) of 4-(N-carbobenzoxy-L-phenylalanylamino)-butyric acid coumarin-7-yl ester for its TGase-mediated hydrolysis and for its enzymatic reaction with the acyl acceptor substrates N-acetyl-L-lysine methyl ester and aminoacetonitrile. We ascertained that the fluorescent substrate was selective toward tissue TGase by testing it with different enzymes, namely microbial transglutaminase (mTGase), Factor XIIIa, papain, and gamma-glutamyl transpeptidase. 4-(N-carbobenzoxyglycinylamino)-butyric acid coumarin-7-yl ester, lacking the benzyl side chain, was also found to be an efficient fluorogenic substrate of tissue TGase. Finally, we have shown that this method is applicable to 96-well microtiter plate format.     

A high-performance liquid chromatographic method for the assay of cholesterol 7 alpha-hydroxylase activity

A high-performance liquid chromatographic method has been developed for the measurement of cholesterol 7 alpha-hydroxylase activity in liver microsomes. 7 alpha-Hydroxycholesterol generated from endogenous cholesterol was derivatized with anthroyl 1-carbonitrile, chromatographed on a reverse-phase column, and detected fluorometrically. The detection limit of 7 alpha-hydroxycholesterol was 1 ng/tube. The cholesterol 7 alpha-hydroxylase activity in rat liver microsomes was assayed by this method, and the effects of some detergents and of the addition of exogenous cholesterol together with detergents on the enzyme activity were investigated. The endogenous 7 alpha- and 7 beta-hydroxycholesterol could be also measured by this method.     

Rapid high-performance liquid chromatographic assay for atovaquone

A rapid high-performance liquid chromatography assay has been developed for the drug atovaquone, which is currently being used to treat Pneumocystis carinii pneumonia and Taxoplasma gondii encephalitis associated with the acquired immunodeficiency syndrome (AIDS). Protein is precipitated from plasma with acetonitrile-aqueous 1% acetic acid (85:15). The supernatant is assayed on a C₆ column using methanol-10 mM triethylamine in aqueous 0.2% trifluoroacetic acid (76:24) with detection at 254 nm. The working assay range was 0.5 to 50 μg/ml. Recovery was 97% and the between-day coefficients of variation were 2.1% at 50 μg/ml and 10.3% at 1 μg/ml. A number of drugs commonly used to treat AIDS and its complications did not interfere with the assay.     

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.     

Stereoselective high-performance liquid chromatographic assay with fluorometric detection for the isomers of mivacurium in human plasma

A high-performance liquid chromatographic assay with fluorometric detection was developed for the analysis of the stereoisomers of mivacurium, a new short-acting neuromuscular blocker, in plasma. The isomers were isolated from plasma by solid-phase extraction with C₁₈ and anion-exchange cartridges. The extracts were chromatographed on a LiChrosphere 60 RP Select B column (125 mm × 4.6 mm I.D.) using a mobile phase of acetonitrile—water (4:6, v/v) containing 0.005 M octanesulfonic acid. The fluorescence excitation and emission wavelengths were 202 and 320 nm, respectively. The accuracy and precision of the assay, expressed as the percentage deviation of measured values from true values and the percentage coefficient of variation, respectively, were ≤ 10% at all concentrations except for the percentage coefficient of variation at the lower limit of quantitation (5 ng/ml). The assay has been successfully used for the analysis of plasma samples from a pharmacokinetic study in human volunteers.     

Thiopurine methyltransferase activity: new high-performance liquid chromatographic assay conditions

This paper reports change to our previously published high-performance liquid chromatographic method for the measurement of 6-methylmercaptopurine (6-MMP) in red blood cell lysates. The extraction procedure and chromatographic conditions have been improved and the range of the calibration curves has been modified. The recoveries of 10 and 100 ng ml⁻¹ 6-MMP were 99.0±6.0% and 96.3±4.0% respectively and the limit of quantification was lowered to 5 ng ml⁻¹. This method, which does not require radioactive S-adenosyl--methionine, is more sensitive, specific and reproducible and may prove useful for routine determination of thiopurine methyltranferase activity in red blood cells.     

Paired-ion high-performance liquid chromatographic assay for plasma choline

Choline was isolated from deproteinized plasma by cation-exchange chromatography. Isolated choline was directly converted to the 3,5-dinitrobenzoate derivative and was analyzed by paired-ion high-performance liquid chromatography with UV detection at 254 nm. An internal standard, 3-hydroxy-N,N,N-trimethylpropanaminium iodide was used for quantitation of plasma choline.Linearity was achieved from 1–500 nmole/ml with a reproducibility of ± 6%. Plasma choline concentrations below 1 nmole/ml could not be accurately measured while plasma choline concentrations in the μmole/ml range deviated from linearity.     

Stereospecific high-performance liquid chromatographic assay of metoprolol

A valid, sensitive high-performance liquid chromatographic technique is reported for the separation of the two enantiomers of metoprolol in human plasma. The procedure involves pre-column derivatization with the homochiral reagent S-(+)-1-(1-naphthyl)-ethyl isocyanate. Once formed, the diastereomers are separated using normal-phase high-performance liquid chromatography. Fluorescence detection (220 nm excitation; no emission filter) was utilized, resulting in baseline resolution (R_s > 1.5). The peaks corresponding to metoprolol enantiomers were free from interference throughout the examined range of 5–500 ng/ml; accuracy and precision were within approximately 10%. Analysis of a plasma sample collected from a healthy volunteer demonstrated that the assay is applicable to clinical studies.     

Thiopurine methyl transferase activity: new extraction conditions for high-performance liquid chromatographic assay

A new liquid–liquid extraction is described for thiopurine methyl transferase (TPMT, EC 2.1.1.67) activity determination: the use of a pH 9.5 NH₄Cl buffer solution, before adding the solvent mixture, allows more rapid extraction, avoiding a centrifugation step, and reduces the global cost of analysis. After the extraction step, 6-methylmercaptopurine, synthesised during the enzymatic reaction, is determined by a liquid chromatographic assay. Analytical performance of the assay was tested on spiked erythrocyte lysates. The linear concentration range was 5–250 ng ml ⁻¹ (r≥0.997, slope=1.497, intercept=−0.367). The recoveries were 82.8, 89.9 and 82.2% for 75, 125 and 225 ng ml⁻¹, respectively. The coefficients of variation were ≤6.1% for within-day assay (n=6) and ≤9.5% for between-day assay precision (n=6; 14 days). TPMT activity was determined in a French adult Caucasian population (n=70). The results ranged from 7.8 to 27.8 nmol h⁻¹ ml⁻¹ packed red blood cells and the frequency distribution histogram is similar to that previously published.     

A fluorometric assay for cholesterol reductase activity.

A fluorometric method for the assay of cholesterol reductase activity from pea leaves (Pisum sativum) is presented. This method is based on the decrease in relative fluorescence occurring as a result of the oxidation of NADH when cholesterol is reduced catalytically to coprostanol by cholesterol reductase. The reaction mixture consisted of micellar cholesterol, NADH, and cytosol of pea leaves in a phosphate buffer. After incubation for 1 h, the reaction mixture were diluted with 2-(N-cyclohexylamino)ethanesulfonic acid buffer (50 mM, pH 10.0) to an appropriate concentration for NADH quantification. The relative fluorescence was measured at an excitation wavelength of 360 nm and at an emission wavelength of 460 nm. This fluorometric method is relatively rapid, simple, and inexpensive. The results obtained show close correlation (R = 0.997) with those obtained by the more time-consuming and expensive radiometric method for assay of cholesterol reductase activity. Results suggest that the fluorometric method is useful for the accurate determination of cholesterol reductase activity in biological specimens.     

A reverse-phase liquid chromatographic assay for flavin-containing monooxygenase activity

A rapid, convenient assay for flavin-containing monooxygenase activity is described. The method is based on direct analysis of quenched incubation mixtures by reverse-phase liquid chromatography, and utilizes p-nitrophenyl-1,3-oxathiolane as the substrate. The synthesis of the substrate and the product are described. The usefulness of p-nitrophenyl-1,3-oxathiolane S-oxide formation as a measure of flavin-containing monooxygenase activity was demonstrated using highly purified and microsomal hog and rat liver flavin-containing monooxygenase. The assay is especially useful for determining stereoselectivity of flavin-containing monooxygenase activity in small amounts of crude tissue preparations.     

Stereoselective high-performance liquid chromatographic assay for propranolol enantiomers in serum

A simple and sensitive stereoselective high-performance liquid chromatographic assay for the quantitation of propranolol enantiomers in serum is described. The method involves conversion of the propranolol enantiomers to diastereomeric urea derivatives by reaction with the chiral reagent (+)-phenylethylisocyanate, followed by chromatographic separation of the diastereomeric products. Conditions of the derivatization reaction were optimized to achieve rapid and quantitative yield with either of the enantiomers. Baseline resolution of the diastereomers was achieved on a reversed phase C₈ column with an isocratic mobile phase. Fluorescence detection afforded an absolute on-column detection limit of 100 pg. The assay has been applied to pharmacokinetic studies in humans and small laboratory animals.     

A fluorometric assay for angiotensin-converting enzyme activity

A simple and sensitive assay for angiotensin-converting enzyme (ACE; EC 3.4.15.1) activity has been developed which employs fluorescently labeled tripeptides. ACE hydrolyzes dansylphenylalanyl-arginyl-tryptophan or dansyl-phenylalanyl-arginyl-phenylalanine, liberating dansyl-phenylalanine and a dipeptide. Dansyl-phenylalanine partitions quantitatively into chloroform, whereas the substrates are virtually insoluble in chloroform. This allows rapid measurement of ACE activity with high signal-to-noise ratios even when microliter aliquots of human serum are assayed. Inhibition studies of the dansyl-tripeptide cleaving activity of human serum and rat lung, the identity of the products of enzyme action, and the regional distribution of enzyme activity among rat tissues demonstrate that only ACE cleaves these substrates under the conditions employed here. This assay may be useful for the clinical measurement of human serum ACE activity and for research investigations of ACE from a variety of tissues.     

Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome

An RP-HPLC assay was developed for a recombinant adenovirus type 5. During chromatography, intact adenovirus dissociated into its structural components (DNA and proteins) and the viral proteome was separated yielding a characteristic fingerprint. The individual components were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, N-terminal sequencing and amino acid composition. The assay was utilized to measure adenovirus particle concentration through quantification of structural proteins. Each structural protein provided independent measurement of virus concentration allowing verification of accuracy. The assay sensitivity is at or below 2·10⁸ particles. Contrary to the benchmark spectrophotometric assay, the RP-HPLC assay was shown to be insensitive to contaminants common for partially purified adenovirus preparations.