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.     

The protein concentration of crude cell and tissue extracts as estimated by the method of dye binding: comparison with the Lowry method.

This paper describes a new, inexpensive, and highly sensitive voltammetric assay for aromatic l-amino acid decarboxylase (AADC) activity in rat and human brains by highperformance liquid chromatography (hplc). l-Dopa was used as substrate and d-dopa for the blank. After isolating dopamine formed enzymatically from l-dopa on a small Amberlite CG-50 column, the dopamine in the column eluate was assayed by hplc with a voltammetric detector. Dihydroxybenzylamine was added to each incubation mixture as an internal standard and therefore this assay was highly reproducible. The peak height in hplc was linear from 100 fmol to 100 pmol of dopamine. The values obtained by this method agreed with those by radioassay using [1-¹⁴C]dopa. The enzyme activity in rat cerebral cortex and in Parkinsonian caudate nucleus, in which the activity was too low to be measured even with the radioassay, could be measured accurately.     

New and highly sensitive assay for l-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography—voltammetry

This paper describes a new, inexpensive and highly sensitive assay for aromatic l-amino acid decarboxylase (AADC) activity, using l-5-hydroxytryptophan (l-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. l-5-HTP was used as substrate and d-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from l-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using l-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.     

New and highly sensitive assay for l-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography—voltammetry

This paper describes a new, inexpensive and highly sensitive assay for aromatic l-amino acid decarboxylase (AADC) activity, using l-5-hydroxytryptophan (l-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. l-5-HTP was used as substrate and d-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from l-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using l-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.     

Highly Sensitive Assay for Dopamine-β-Hydroxylase Activity in Human Cerebrospinal Fluid by High Performance Liquid Chromatography-Electrochemical Detection: Properties of the Enzyme

Abstract: This paper describes a new, sensitive assay for dopamine-β-hydroxylase (DBH) activity in human cerebrospinal fluid (CSF), serum and brain tissues by high performance liquid chromatography (HPLC) with electrochemical detection (ED). Dopamine (DA) was used as a substrate and was incubated under optimal conditions. Norepinephrine (NE) formed enzymatically from DA was isolated by a double-column procedure, the first column of Dowex-50-H⁺ and the second column of aluminum oxide. NE was adsorbed on the second aluminum oxide column and then eluted with 0.5 M-hydrochloric acid and assayed by HPLC-ED. Epinephrine (EN) was added to each incubation mixture as an internal standard, and this assay was therefore highly reproducible. The peak height in HPLC was linear from 500 fmol to 100 pmol of NE and EN. The lower limit of detection for NE formed enzymatically was about 30 pmol, which indicated that the sensitivity of this procedure was comparable to that of radioassay procedures. We applied the method to measurement of the activity of and examination of some of the characteristics of DBH in human CSF. DBH activity in CSF of Parkinsonian patients was lower than that of control patients. The properties of DBH in human CSF were similar to those in serum and adrenal medulla.     

A modified radioisotopic assay for measuring glutamine synthetase activity in tissue extracts.

A radioisotope assay for the measurement of glutamine synthetase activity has been developed in which tandemly arranged ion-exchange columns of Dowex 1-acetate and Amberlite CG-50 (H⁺) are used to separate the product, [¹⁴C]glutamine, from unreacted [U-¹⁴C]glutamate and other labeled compounds, particularly γ-aminobutyrate, that are formed by competing reactions. The technique is sensitive, reproducible, and suitable for multiple determinations. The assay has been used successfully to measure glutamine synthetase activity in neural and nonneural tissues which contain appreciable amounts of glutamate decarboxylase activity.     

High-performance liquid chromatographic quantification of 4-methylumbelliferyl-β-d-glucuronide as a probe for human β-glucuronidase activity in tissue homogenates

An internally standardized HPLC method to determine the concentration of 4-methylumbelliferone liberated from 4-methylumbelliferyl-β-d-glucuronide by human β-glucuronidase was developed. The assay allows the precise and rapid measurement of specific enzyme activity in human tissue homogenates. Without prior extraction the incubation mixture can be separated using a C₈ column followed by fluorescence detection. The assay showed good accuracy and precision with a detection limit of 20 nM and a limit of quantification of 167 nM. The suitability of the method was shown in enzyme kinetic experiments with human liver homogenates.     

Author index for volume 84

A rapid method for hemoglobin chain recombination which gives a homogeneous product was developed. The method utilizes a small carboxymethylcellulose column as a medium for chain recombination and concentration of the hemoglobin. Equimolar amounts of p-hydroxymercuribenzoate derivatives of α- and β-chains were mixed with 300× molar excess of β-mercaptoethanol over the p-hydroxy mercuribenzoate groups. After 10 min of incubation in an ice bath, the mixture was adjusted to pH 5.85, and was loaded on a carboxymethylcellulose column. The column was washed with 10 mm phosphate buffer-1 mm Na₂EDTA-47 mm β-mercaptoethanol, pH 5.85 and then with 10 mm phosphate buffer, pH 5.85. The hemoglobin was eluted from the column by use of 15 mm K₂HPO₄. The hemoglobin was homogeneous on polyacrylamide gel electrophoresis and had a visible spectrum, electrophoretic mobility, and number of -SH groups comparable to those shown by control hemoglobin.     

Homogeneous cytochrome b5 from human erythrocytes

Homogeneous cytochrome b₅ has been isolated from large volumes of human erythrocytes by sequential chromatography on DEAE-cellulose, Amberlite CG-50, Bio-Gel P-60, and DEAE-Sephadex A-50. A molecular weight of 15,300 was determined by SDS disc gel electrophoresis. Trypsin converted the protein to a smaller hemepeptide which was indistinguishable from trypsin-cytochrome b₅ of human liver microsomes by disc gel electrophoresis. The data suggest that erythrocyte cytochrome b₅ has the same structure as a segment of liver microsomal cytochrome b₅ and is intermediate in size between the trypsin- and detergent-solubilized forms of the liver protein.     

Dephosphorylation of purified brain tyrosine hydroxylase by rat striatal extracts

Tyrosine hydroxylase (TH) was purified from bovine brain and enzymatically phosphorylated in vitro. Radioactively phosphorylated TH was dephosphorylated by rat tissue extracts. Of tissues examined, rat corpus striatal extracts were highest in specific activity in the TH dephosphorylating assay. Phosphorylated histone did not inhibit dephosphorylation of TH by rat striatal extracts. The thermal decay of dephosphorylating activity of rat striatal extracts varied with substrate, with TH dephosphorylating activity most unstable of the activities assayed. The results suggest that TH can be enzymatically dephosphorylated and that, in corpus striatum, this process differs quantitatively from the dephosphorylation of phosphohistone and phosphoprotamine.     

Phylogenetically Distinct Bacteria Involve Extensive Dechlorination of Aroclor 1260 in Sediment-Free Cultures

Microbial reductive dechlorination of the persistent polychlorinated biphenyls (PCBs) is attracting much attention in cleanup of the contaminated environment. Nevertheless, most PCB dechlorinating cultures require presence of sediment or sediment substitutes to maintain their dechlorination activities which hinders subsequent bacterial enrichment and isolation processes. The information on enriching sediment-free PCB dechlorinating cultures is still limited. In this study, 18 microcosms established with soils and sediments were screened for their dechlorination activities on a PCB mixture – Aroclor 1260. After one year of incubation, 10 out of 18 microcosms showed significant PCB dechlorination with distinct dechlorination patterns (e.g., Process H, N and T classified based on profiles of PCB congeners loss and new congeners formation). Through serial transfers in defined medium, six sediment-free PCB dechlorinating cultures (i.e., CW-4, CG-1, CG-3, CG-4, CG-5 and SG-1) were obtained without amending any sediment or sediment-substitutes. PCB dechlorination Process H was the most frequently observed dechlorination pattern, which was found in four sediment-free cultures (CW-4, CG-3, CG-4 and SG-1). Sediment-free culture CG-5 showed the most extensive PCB dechlorination among the six cultures, which was mediated by Process N, resulting in the accumulation of penta- (e.g., 236-24-CB) and tetra-chlorobiphenyls (tetra-CBs) (e.g., 24-24-CB, 24-25-CB, 24-26-CB and 25-26-CB) via dechlorinating 30.44% hepta-CBs and 59.12% hexa-CBs after three months of incubation. For culture CG-1, dechlorinators mainly attacked double flanked meta-chlorines and partially ortho-chlorines, which might represent a novel dechlorination pattern. Phylogenetic analysis showed distinct affiliation of PCB dechlorinators in the microcosms, including Dehalogenimonas and Dehalococcoides species. This study broadens our knowledge in microbial reductive dechlorination of PCBs, and provides essential information for culturing and stimulating PCB dechlorinators for in situ bioremediation applications.     

Assay of L-aromatic amino acid decarboxylase by high performance liquid chromatography

A method is presented for the assay, by high-performance liquid chromatography, of l-aromatic amino acid decarboxylase. The K_m value of the enzyme for 3,4-dihydroxyphenylalanine (dopa) was, by this procedure, 0.16 mm, in good agreement with previous reports. When α-methyldopa was used as substrate, evidence was obtained indicating the formation of, besides α-methyldopamine, 3,4-dihydroxyphenylacetone, presumably through an internal transamination process.     

Structural analysis and antioxidant activity of the glycoside from Imperial Chrysanthemum

A new glycoside, named as CG-1, was separated from Imperial Chrysanthemum with silica gel column chromatography. The purity was detected by TLC and HPLC. The crystal shape of CG-1 was consisting of a quadrangular and two rectangular pyramids. The analysis of DSC and TGA showed that the melting point of CG-1 crystal was 150.22?°C and had good thermal stability. The monosaccharide conformation analysis showed that it was d-glucose. The structure characteristics were compared by FT-IR, NMR spectroscopy and LC-MS and a molecular structure has been deduced which consistent with spectroscopic results. In vitro antioxidant results suggested that the glycoside extracted from Imperial Chrysanthemum could be effectively employed as natural antioxidant in health or functional food. This work is of significance to keep the antioxidant activity in the processing and application of Imperial Chrysanthemum.     

Studies on glutathione-S-arene oxidase transferase. A sensitive assay and partial purification of the enzyme from sheep liver

A sensitive assay has been devised for glutathione-S-arene oxidase transferase using as substrates naphthalene-1,2-oxide or styrene oxide along with [³⁵S]glutathione. Activity of the order of 2–3 nmoles of conjugate formed during a 5-min incubation can be detected. This yields about 2000 cpm above a blank of about 1500 cpm. Transferase activity was found mainly in liver and kidney but was also present in most other tissues of rats. Glutathione-S-arene oxide transferase has been purified 70- to 80-fold from sheep liver 100,000 g supernatants using the conventional procedures. After electrofocusing, enzyme activity separated into two major peaks and two or three minor peaks, ranging in isoelectric point from pH 6.5 to 7.5. Activities assayed with naphthalene-1,2-oxide or styrene oxide as substrates were found to almost parallel each other in all the peaks.The sheep liver transferase required neither metal ions nor cofactors such as FAD, pyridoxal-phosphate and thiamine pyrophosphate. The molecular weight of the transferase has been estimated to be about 40,000.K_m values for glutathione, naphthalene-1,2-oxide, and styrene oxide are 1.6, 0.11, and 0.13 mm, respectively. K_m values for glutathione decreased with increasing pH, whereas the K_m values for naphthalene-1,2-oxide were independent of pH in the range of 6.5–8.     

Coupling of glucose oxidase and Fenton's reaction for a simple and inexpensive assay of β-glucosidase

A simple and inexpensive assay for β-glucosidase, based on the coupling of glucose oxidase and Fenton's reagent has been described. Hydrogen peroxide formed as a result of the action of glucose oxidase on glucose (derived from the action of β-glucosidase on cellobiose) oxidizes ferrous sulphate, resulting in an increase in absorbance. The oxidation products produced a peak of maximum absorbance at 340 nm. Using this assay system, a linear relationship between glucose concentration in the range 5.55–27.78 mmol l^?1(100–500 mg dl^?1) and absorbance was obtained, indicating conformity to Beer's law. The preciseness of the glucose oxidase/Fenton's reagent for the assay of glucose was shown to be satisfactory. β-Glucosidase was assayed using the hexokinase assay reagent and the glucose oxidase/ferrous sulphate reagent. The values obtained using both reagents did not differ significantly. Although 2.6 times less sensitive than the hexokinase reagent when absorbance is measured at 340 nm, the glucose oxidase/Fenton's reagent is 10 times cheaper and could be used satisfactorily for routine assays of β-glucosidase and other carbohydrases including cellulase and amylase. In this respect, fructose, mannose, xylose, sucrose and cellobiose did not affect the sensitivity of the reagent. Of several metals tested, only aluminium interfered with the reagent, decreasing its sensitivity.     

A new procedure for the purification of rat testis-specific histone TH2B involving affinity-related chromatography

A new procedure for the isolation of rat testis-specific histone TH2B has been devised. First, rat testis chromatin fragments were applied to a hydroxylapatite column in 0.5 m NaCl, 0.1 m potassium phosphate buffer, pH 6.7, and histones were selectively stripped off the bound DNA in groups (H1/TH1, H2A/H2B/TH2B, and H3/H4). The fraction containing H2A, H2B, and TH2B, but lacking H3, was reduced, desalted, and applied to a p-chloromercuribenzoyl-aminoethyl-Sepharose 4B-CL column in 8 m urea, 0.1 m Tris-HCl, pH 8.1. After washing with the same buffer to remove H2A and H2B, covalently bound TH2B was eluted out with 10 mm dithiothreitol in the same buffer. No contaminants were detectable in the purified TH2B either by polyacrylamide gel electrophoresis in 0.4% Triton X-100, 2.5 m urea, 0.9 n acetic acid, or by N-terminal analysis with dansyl chloride.     

The effects of phosphorylating conditions on tyrosine hydroxylase activity are influenced by assay conditions and brain region

Using the supernatant fraction of rat brain homogenates, we investigated several variables which appear to be important in studies of tyrosine hydroxylase (TH) activity. These included the type and pH of the assay buffer, cofactor concentration, and brain region. We observed that the pH optimum for TH activity assayed in Tris-acetate buffer varied with brain region. Among the regions examined, the optima ranged from pH 5.7 (striatum) to pH 6.2 (hippocampus). Similar results were obtained using MES buffer, although TH activity was reduced at certain pH values. The pH optimum was not correlated with the relative proportions of norepinephrine and dopamine in these brain regions. In the presence of a subsaturating concentration of cofactor, incubation of TH under cAMP-dependent protein phosphorylating conditions increased TH activity significantly in both striatum and hippocampus. The increase in TH activity produced by phosphorylating conditions was most pronounced at pH values above the pH optimum. The results are discussed in terms of their implications for $\text{in vitro}$ measurement of alterations in TH activity.     

Hydrophobic-ionic chromatography: its application to microbial glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase

Glucose oxidase from Aspergillus niger, hyaluronidase from Streptomyces hyalurolyticus, and cholesterol oxidase and cholesterol esterase from Pseudomonas fluorescens were effectively adsorbed on an Amberlite CG-50 column, when the cell-free cultured medium or the cultured medium with cell extract and without cell debris was applied without desalting but at pH less than or equal to 4.5. At the acidic pH, all the ion-exchange groups (-COOH) exist in the protonated form; the adsorption is not due to electrostatic attraction, but to hydrophobic interaction. The enzymes thus adsorbed were effectively eluted by increasing pH, at which the ion-exchange groups became dissociated. This type of adsorption-elution is called hydrophobic-ionic chromatography. By a single run of chromatography, glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase were purified 30-fold, 12-fold, 45-fold, and 20-fold with yields of 82%, 83%, 80%, and 90%, respectively. This indicates that hydrophobic-ionic chromatography on an Amberlite CG-50 column is effective for the purification of various enzymes, provided that they are stable at the acidic pH.     

An automated assay for brain serotonin

This communication describes an automated assay for brain serotonin. The assay is an adaptation of a commonly used manual assay which utilizes the reaction of o-phthalaldehyde with serotonin to develop fluorescence (2). In the automated assay the samples to be analyzed were mixed with 7.5 N HCl and o-phthalaldehyde in the presence of reduced glutathione, heated at 80°C, cooled and their fluorescence recorded. Linearity with respect to serotonin was demonstrated from 0 to at least 5 μM, and the lower limit of sensitivity was 0.7 ng serotonin (0.4 ml of 0.01 μM). The % error (standard deviation times 100 divided by the mean) was always 2% or less. The specificity was studied, and the assay was applied to the measurement of rat brain serotonin levels by demonstrating an increase in brain serotonin levels after pargyline treatment and a decrease after reserpine treatment.