ABSTRACT
Using spectroscopic techniques we studied the effect of the nucleophilic reagents cyanide, cyanate and thiocyanate on three flavo-oxidases namely alcohol oxidase (AO), glucose oxidase (GOX) and D-amino acid oxidase (DAOX). All three ions, added at concentrations in the mM range, caused release of the flavin adenine dinucleotide (FAD) co-factors from the enzyme molecules. In the case of AO this was accompanied by significant conformational perturbations, which was not observed for GOX and DAOX. As suggested from fluorescence, absorption and circular dichroism spectral changes at least one phenolic hydroxyl group became ionized upon FAD release from AO and a new class of Trp residues, fluorescent only in apo-AO protein, was demasked.
Subject(s)
Flavin-Adenine Dinucleotide/chemistry , Flavoproteins/chemistry , Oxidoreductases/chemistry , Alcohol Oxidoreductases/chemistry , Circular Dichroism , Cyanates , Cyanides , D-Amino-Acid Oxidase/chemistry , Glucose Oxidase/chemistry , Indicators and Reagents , Spectrometry, Fluorescence , Spectrophotometry , ThiocyanatesABSTRACT
The biologically active form of interferon gamma (IFN-gamma) is a dimer consisting of two identical non-covalently bound polypeptide chains. We have studied spectroscopically the dimer-monomer dissociation equilibrium of human recombinant IFN-gamma and have found that the monomers possess approximately 50% lower Trp quantum yield than the dimers [Boteva et al. Biochemistry 1996;35:14825]. In the present study we characterise the conformational properties of the two states--monomeric and dimeric, and analyse the effects of the salt composition of human blood plasma, physiological cations K+, Na+, Ca2+ and Mg2+ and mechanical stress on the dimer-monomer equilibrium. A medium with electrolyte composition of human blood plasma increases both the association and dissociation rate constants without shifting significantly the dimer-monomer equilibrium. The physiological cations shift the equilibrium towards dissociation of dimers into monomers by lowering the activation energy and the free energy of the process thus decreasing the stability of IFN-gamma. Mechanical stress caused by stirring of the protein solution reduces irreversibly the Trp fluorescence by 75-80% and decreases significantly the alpha-helical content and favours the aggregation.
Subject(s)
Interferon-gamma/chemistry , Acrylamides , Cations, Monovalent , Dimerization , Humans , Kinetics , Metals , Protein Structure, Secondary , Recombinant Proteins , Spectrometry, Fluorescence , TryptophanABSTRACT
The biologically active form of interferon gamma is a dimer composed of two noncovalently bound identical polypeptide chains of 17 kDa each. In this study, it was found that dissociation of the dimer into monomers significantly reduced the fluorescence quantum yield and the efficiency of the intermolecular Tyr to Trp radiationless energy transfer. The same process caused significant changes in the fluorescence decay and in the fluorescence anisotropy decay. The kinetic and thermodynamic parameters of the dimer-monomer equilibrium were determined by fluorescence measurements at different temperatures and by a theoretical mathematical model. Dissociation of the dimers into monomers was an endothermic process and was favored by concentrations of the protein lower than 1 microM and by increasing the temperature. It was accompanied by formation of aggregates, a slow and partially reversible process leading to inactivation of the interferon. It is suggested that certain monomeric conformers are competent for aggregation.
Subject(s)
Interferon-gamma/chemistry , Biopolymers/chemistry , Fluorescence Polarization , Humans , Mathematics , Protein Conformation , Recombinant Proteins , Spectrometry, Fluorescence , Thermodynamics , Tryptophan/chemistry , Tyrosine/chemistryABSTRACT
Arthropod hemocyanin (isolated from the crab Carcinus maenas and the lobster Homarus americanus) is usually referred to as an oxygen transport-storage protein. The protein, however, also catalyses with low efficiency the oxidation of o-diphenol to quinone, similarly to tyrosinase (monophenol, o-diphenol:oxygen oxidoreductase). The enzymatic parameters of hemocyanin are affected by the aggregation state of the protein; namely V(max) exhibited by a dissociated subunit is one order of magnitude greater than that of aggregated species. The reaction velocity is increased by the presence of perchlorate, an anion of the Hofmeister series. The results are also discussed on the basis of active site accessibility in comparison with tyrosinase.
Subject(s)
Brachyura/chemistry , Catechol Oxidase/metabolism , Hemocyanins/metabolism , Nephropidae/chemistry , Animals , Catechols/metabolism , Hemocyanins/chemistry , Hemocyanins/isolation & purification , Monophenol Monooxygenase/metabolism , Oxidation-Reduction , Perchlorates/chemistry , Perchlorates/pharmacology , Protein Conformation , Quinones/metabolism , Sodium Compounds/chemistry , Sodium Compounds/pharmacology , Time FactorsABSTRACT
The kinetic properties dextran-chymotrypsin conjugate were studied by means of low molecular weight substrates. It was found that KM, kcat and kcat/KM of dextran chymotrypsin for the hydrolysis of benzoyl-L-tyrosine-ethyl-ester did not differ substantially from those of the free enzyme. However, the data found for kcat of dextran-chymotrypsin and free chymotrypsin assayed for the hydrolysis of three tripeptidyl-p-nitroanilide D-Arg-Val-Trp-pNA, D-Arg-Val-Tyr-pNA, Z-Phe-Pro-Phe-pNA, were definitely different. The inhibition of the modified chymotrypsin with soybean trypsin inhibitor was found to be less pronounced than that with the free enzyme. The effect of potassium and magnesium salts on the inactivation of both enzymes was also studied. The effect of dextran matrix on the catalytic properties and the conformational stability of modified chymotrypsin is discussed.