Laccase assay by means of high-performance liquid chromatography

Spectrophotometric determination of laccase activity may be affected by the formation of quinoid chromophores arising from nonenzymatic oxidations interfering with enzymatic reactions. Km values for guaiacol obtained by spectrophotometric and HPLC methods confirm the above hypothesis. HPLC results are particularly useful for the assay of laccase activity on natural phenolic extracts.     

Sensitive high-performance liquid chromatographic assay for pilocarpine in biological fluids using fluorescence derivatisation

A sensitive assay for pilocarpine in biological fluids has been developed involving HPLC of a fluorescent derivative of 4-bromomethyl-7-methoxycoumarin. Pilosine as internal standard was added before the derivatisation step. The fluorescent derivatives were well resolved and separated from excess reagent and endogeneous compounds on a cyanopropyl silica column. The detection limit of pilocarpine in biological fluids was 1.0 ng/ml and the assay was linear up to a concentration of 150 ng/ml. The assay was applied to a preliminary study of pilocarpine disposition in man after a single oral dose. This is the first report of pilocarpine excretion into saliva.     

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.     

Natural substrate assay for chitinases using high-performance liquid chromatography: a comparison with existing assays

The determination of kinetic parameters of chitinases using natural substrates is difficult due to low K(m) values, which require the use of low substrate concentrations that are hard to measure. Using the natural substrate (GlcNAc)(4), we have developed an assay for the determination of k(cat) and K(m)values of chitinases. Product concentrations as low as 0.5 microM were detected using normal-phase high-performance liquid chromatography (HPLC) with an amide 80 column (0.20 x 25 cm) using spectrophotometric detection at 210 nm. By means of this assay, k(cat) and K(m)values for chitinases A (ChiA) and B (ChiB) of Serratia marcescens were found to be 33+/-1s(-1) and 9+/-1 microM and 28+/-2s(-1) and 4+/-2 microM, respectively. For ChiB, these values were compared to those found with commonly used substrates where the leaving group is a (nonnatural) chromophore, revealing considerable differences. For example, assays with 4-methylumbelliferyl-(GlcNAc)(2) yielded a k(cat) value of 18+/-2s(-1) and a K(m) value of 30+/-6 microM. For two ChiB mutants containing a Trp --> Ala mutation in the +1 or +2 subsites, the natural substrate and the 4-methylumbelliferyl-(GlcNAc)(2) assays yielded rather similar K(m) values (5-fold difference at most) but showed dramatic differences in k(cat) values (up to 90-fold). These results illustrate the risk of using artificial substrates for characterization of chitinases and, thus, show that the new HPLC-based assay is a valuable tool for future chitinase research.     

A high-performance liquid chromatography assay for Dyrk1a,a Down syndrome-associated kinase

Down syndrome is the most common aneuploidy. It is caused by the presence of an extra copy of chromosome 21. Several studies indicate that aberrant expression of the kinase Dyrk1a (dual-specificity tyrosine phosphorylation-regulated kinase 1a) is implicated in Down syndrome, in particular in the onset of mental retardation. Moreover, elevated Dyrk1a activity may also be a risk factor for other neurodegenerative disorders such as Alzheimer’s disease. Over the past years, Dyrk1a has appeared as a potential drug target. Availability of sensitive and quantitative enzyme assays is of prime importance to understand the role of Dyrk1a and to develop specific inhibitors. Here, we describe a new method to measure Dyrk1a activity based on the separation and quantification of specific fluorescent peptides (substrate and phosphorylated product) by high-performance liquid chromatography (HPLC). Kinetic and mechanistic analyses using well-known inhibitors of Dyrk1a confirmed the reliability of this approach. In addition, this assay was further validated using brain extracts of mice models expressing different copies of the Dyrk1a gene. Our results indicate that this novel Dyrk1a assay is simple, sensitive, and specific. It avoids the use of radioactivity-based approaches that, until now, have been widely employed to measure Dyrk1a activity.     

Determination of glucosamine in impure chitin samples by high-performance liquid chromatography

A simple, rapid, selective, and specific high-performance liquid chromatography (HPLC) method was developed to quantitate glucosamine, and its application for estimating purity of chitin was investigated. The chromatographic separation was achieved using a reversed-phase C8 column, pre-column derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) and ultraviolet detection (lambda=254 nm). The mobile phase consisted of CH3CN and H2O. The optimum conditions of acid hydrolysis of chitin (concentration of HCl, temperature, and heating time) was obtained by performing the orthogonal array design (OAD) procedure and the released glucosamine was determined by the above HPLC method. The accuracy of the method was checked by the standard addition technique. The method was found to be specific with good linearity, accuracy, precision, and well suited for quantitation of glucosamine and determination of the purity of chitin in biological materials and food products.     

Determination of alendronate in human urine as 9-fluorenylmethyl derivative by high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantitation of alendronate as the 9-fluorenylmethyl derivative (FMOC) in human urine is presented. The sample preparation involved coprecipitation with calcium phosphate, separation on diethylamine (DEA) solid-phase extraction (SPE) cartridge and derivatization with 9-fluorenylmethyl chloroformate in citrate buffer pH 11.9. Liquid chromatography was performed on an octadecylsilica column (150 x 4.6 mm, 3 microm particles); a gradient method with starting mobile phase acetonitrile-methanol-citrate/pyrophosphate buffer (20:15:65 v/v) was employed. The total run time was 21 min. The fluorimetric detector was operated at the following wavelengths: 260 nm (excitation) and 310 nm (emission). Pamdronate was used as the internal standard. The limit of quantitation was 3.5 ng/ml using 5 ml of urine. Within-day and between-day precision expressed by relative standard deviation was less than 8% and inaccuracy did not exceed 9%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Purification of fatty acid methyl esters by high-performance liquid chromatography

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane–isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.     

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 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.     

Histone fractionation by high-performance liquid chromatography

A method for the rapid chromatography of histones by high-performance liquid chromatography (HPLC) using a reverse-phase μBondapak C₁₈ column containing a packing of octadecylsilane chemically bonded to silica and a linear elution gradient running from water to acetonitrile is described. Two conditions were found to be necessary to achieve histone fractionation: (i) silylation of the active groups of the silica solid support, and (ii) trifluoroacetic acid (TFA) in the eluting solvents. Greater than 90% of the total [³H]lysine-labeled protein applied to the column was eluted from the column. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The HPLC histone fractions (identified by their electrophoretic mobilities) were eluted from the column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A + H4, (LHP)H3, and (MHP)H3 (where LHP and MHP refer to the less hydrophobic and more hydrophobic histone variants). Phosphorylated histone species were not resolved from their unmodified parental species. The volatile nature of the water/acetonitrile/TFA eluting solvent facilitated the recovery of salt-free histones from the eluted HPLC fractions by simple lyophilization. This system is very useful for the rapid isolation of the lysine-rich histones, H1 and H2B, and the variants of histone H3. With further development, this system is expected to extend the advantages of HPLC to the fractionation of histone H4 and the variants of histone H2A as well.     

Analysis method for lipoproteins by high-performance liquid chromatography with sulfopropyl-ligand column and magnesium ion-containing eluents

We have developed a new analysis method for lipoproteins in serum by high-performance liquid chromatography using a sulfopropyl-ligand column with eluents containing magnesium nitrate. The magnesium ion anchors lipoproteins to the ligands on the column gel. Lipoproteins are eluted from the column with a magnesium nitrate concentration gradient and detected by postcolumn reaction using a reagent containing cholesterol esterase and cholesterol oxidase. High-density lipoprotein, low-density lipoprotein, and very-low-density lipoprotein were eluted in order from the column. The within-assay and between-assay coefficients of variation for cholesterol concentration in lipoproteins were 1.1-3.7 and 1.3-5.8%, respectively. The correlation coefficients between the values of total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol obtained by the new method and those obtained by an enzymatic method using an automated chemical analyzer were 0.940, 0.979, and 0.909, respectively. The new method was successfully applied to the analysis of plasma lipoproteins of patients with hyperlipidemia.     

Direct assay for creatine kinase by reversed-phase high-performance liquid chromatography

A direct assay for creatine kinase (CK) activity was developed based on the separation and quantitation of adenosine triphosphate (ATP) by high-performance liquid chromatography. The total incubation time is 13 min and the elution time for ATP is 16 min. Using lyophilized CK as the sample, a sensitivity in the range of 8 U/l (units/liter) was obtained. The method presented also has clinical significance in that CK levels in serum can easily be determined with minimal sample preparation. Using serum samples from a healthy patient and a heart attack victim, activities of 26.6 U/l and 609.0 U/l, respectively, were obtained. Because of the direct measurement of ATP, this method eliminates the coupled reactions encountered in the common spectrophotometric and colorimetric methods of analysis resulting in a simpler and inexpensive assay.     

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 specific high-performance liquid chromatography assay for dehydroascorbic acid shows an increased content in CLL lymphocytes

A method for the assay of dehydroascorbic acid using high-performance liquid chromatography with uv detection is described. The dehydroascorbic acid is separated from ascorbic acid and reduced with dithiothreitol, and is then quantitated as ascorbic acid following rechromatography. Since as little as 22 pmol can be detected, sensitivity is at least 40-fold greater than that of other currently available procedures. This method was used to measure the level of dehydroascorbic acid in normal and chronic lymphocytic leukemia lymphocytes. A significantly higher concentration of dehydroascorbic acid was found in leukemic (21.80 +/- 3.55 nmol/10(8) cells, mean +/- SE) than in normal lymphocytes (9.32 +/- 1.15 nmol/10(8) cells) (P less than 0.03). Analysis of extracts from normal B cell lymphocytes revealed comparable dehydroascorbic acid levels to unfractionated lymphocytes, indicating that the elevated level in chronic lymphocytic leukemia was not simply a reflection of the increased percentage of B lymphocytes in this disorder. These studies illustrate that the technique can be used to measure the dehydroascorbic acid content from sources where only scanty material is available or low levels are found.     

Assay of galactosyltransferase by high-performance liquid chromatography

Galactosyltransferase catalyzes transfer of galactose from UDP-galactose to glucose or N-acetylglucosamine with resultant formation of galactosides and UDP. In this new assay galactosyltransferase activity is measured by determining UDP by isocratic high-performance liquid chromatography on an amino-bonded column monitored spectrophotometrically. Concurrently, unreacted UDP-galactose and breakdown products arising from UDP-galactose (UMP and uridine) are also determined. The new technique does not require radioactive substrates, permits usage of saturating concentrations of UDP-galactose, and provides monitoring of side reactions.     

Determination of diaminopimelic acid in rat feces by high-performance liquid chromatography using the Pico Tag method

The purpose of the study was to develop a method for the determination of diaminopimelic acid (DAPA) concentrations in rat feces by reversed-phase high-performance liquid chromatography (HPLC) using the Pico Tag method. Precolumn derivatization with phenylisothyocyanate (PITC) and UV (254 nm filter) detection were used. Samples were hydrolysed in 6 M HCl at 110 degrees C for 24 h. Hydrolysates were then diluted, dried and derivatized, and samples (10 microliter injected onto a 300x3.9 mm NovaPak C(18) (Waters) HPLC column. Under the conditions used, DAPA eluted as one single peak between those of tyrosine and valine. On-column DAPA concentrations in standards were 41.5-83 pmol, which were in the range of the amounts present in fecal samples of rats fed semisynthetic diets. Amounts of DAPA determined in fecal samples of rats fed broad bean- or chickpea-based diets were, respectively, 2.56 and 2.98 mg g(-1). The advantages of the method and the relevance of the results for nutritional studies in monogastric animals are discussed.     

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 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.     

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.