ABSTRACT
A high-performance liquid chromatographic method has been developed and validated for the fingerprinting (profiling) and quantitative determination of E- and Z-guggulsterones, the hypolipidemic agents in the gum-resin exudate of Commiphora mukul, currently marketed worldwide as hypocholesterolemic. The method involves extraction of the guggul-resin from either the raw exudate or compounded tablets (or capsules) with ethyl acetate, concentration of the combined extracts and chromatography on a reversed-phase C18 column using an acetonitrile-water gradient. The method has a validated quantitation range of 15-85 microg/ml for E-guggulsterone and 25-130 microg/ml for Z-guggulsterone with a precision of +/-2% S.D. and a recovery of >99.5%. Standard curve correlation coefficients of 0.992 or greater were obtained during validation experiments. The method was applied to six commercial (OTC) products, all of which were found to contain significantly less (in most cases very little or none) of the claimed guggulsterones.
Subject(s)
Chromatography, High Pressure Liquid/methods , Hypolipidemic Agents/analysis , Plants, Medicinal/chemistry , Pregnenediones/analysis , Isomerism , Reproducibility of Results , Spectrophotometry, UltravioletABSTRACT
The amino acid sequence of the neutral zinc protease from Bacillus mesentericus strain 76 (MCP 76) has been determined by using peptides derived from digests with trypsin, chymotrypsin, and cyanogen bromide and from cleavage with o-iodosobenzoic acid. The peptides were purified by means of gel filtration and reversed-phase high-performance liquid chromatography and analyzed by automatic sequencing. The protein contains 300 amino acid residues. It proved to be identical with the neutral protease deduced from the DNA precursor sequence of Bacillus subtilis. The residues for zinc and substrate binding are conserved, whereas the number of calcium binding sites is reduced compared to thermolysin. A classification of the neutral zinc protease is discussed.
Subject(s)
Bacillus/enzymology , Metalloendopeptidases , Amino Acid Sequence , Carboxypeptidases , Carboxypeptidases A , Chromatography, Gel , Chromatography, High Pressure Liquid , Chymotrypsin , Iodobenzoates , Molecular Sequence Data , Peptide Fragments , TrypsinABSTRACT
The milk-clotting protease from Bacillus mesentericus strain 76 is free of carbohydrate and lacks cysteine and cystine. The amino acid composition indicates a single peptide chain with 304 residues. Five amino acids--aspartic acid, threonine, serine, glycine and alanine--represent one-half of the total residues. The enzyme contains 35 aromatic amino acids and 103 ionic amino acids. The observed constant value of the ratio Menzyme:Azinc is 1:1 for active and acid denatured enzyme preparations, indicating that 0.1 M acetic acid causes denaturation of the enzyme but it can not eliminate the zinc ions.
Subject(s)
Aspartic Acid Endopeptidases , Bacillus/enzymology , Endopeptidases/isolation & purification , Amino Acids/analysis , Carbohydrates/analysis , Spectrophotometry, Ultraviolet , Zinc/analysisABSTRACT
Pure milk-clotting protease (MCP-76) is isolated by isotachophoresis at pH 5.0. The native molecule has only one protein chain. It is a metaloenzyme containing zinc. The pure MCP-76 has a molecular weight of 33 000 (+/- 1500) and by diphenyl-indenonyl-isothiocyanate method showed arginine as N-terminal amino acid.
Subject(s)
Aspartic Acid Endopeptidases , Bacillus/enzymology , Endopeptidases/isolation & purification , Milk , Animals , Aspergillus/isolation & purification , Cattle , Molecular WeightABSTRACT
A two-step procedure for the isolation of pure enzyme from Bacillus mesentericus strain 76 is developed. Ion-exchange chromatography on CM-Sephadex-C50 is used as a second step after precipitation from ethanol. The pure enzyme preparation, obtained after gel filtration on Sephadex-G25 of the ion-exchange chromatographically separated product, possess the highest specific activity achieved. The homogeneity of the final preparation is proved by determination of the N-terminal amino acid (Arg) and by SDS-PAGDE (single disc).
Subject(s)
Bacillus/enzymology , Peptide Hydrolases/isolation & purification , Animals , Cattle , Dairy Products , MilkSubject(s)
Bacillus/enzymology , Esterases/metabolism , Peptide Hydrolases/metabolism , Animals , Hydrogen-Ion Concentration , Kinetics , MilkSubject(s)
Plant Proteins , Amino Acids/analysis , Autoanalysis , Buffers , Chemical Phenomena , Chemistry , Chromatography, Gel , Dextrans , Drug Stability , Edible Grain/analysis , Electrochemistry , Electrophoresis , Electrophoresis, Disc , Hydrogen-Ion Concentration , Methods , Molecular Weight , Osmolar Concentration , Plant Proteins/analysis , Plant Proteins/classification , Solubility , UreaSubject(s)
Glucosamine/analysis , Hexosamines/analysis , Amines , Chromatography, Thin Layer , Naphthalenes , SulfurABSTRACT
Dithiocarbaminoacetic acid (TCA) forms very stable, water soluble complexes with a number of metal ions and is a suitable masking agent in complexometry. TCA masks from EDTA and complexometric indicators at pH 2-6 the following elements: bismuth(III), indium(III), thallium(III), cadmium(II), lead(II), mercury(II) and copper(II), thus making possible the complexometric determination of other elements in their presence.
