ABSTRACT
Extrapolating the recent progress in the near future the extensive utilization of cofactor-dependent enzymes (enzymes of the 3rd generation) for solving economic or medical problems will be restricted by the difficulties of cofactor regeneration. Real possibilities exist in analytical systems, for instance enzyme electrodes. In the present paper a special case of overcoming the cofactor regeneration in P-450 catalyzed substrate hydroxylation is demonstrated: The peroxide-dependent reaction gives the same products as obtained under physiological conditions; that is why in an electro-enzyme-reactor producing hydrogen peroxide by cathodic oxygen reduction a considerable simplification of the multi-enzyme complex is possible by omitting electron transfer proteins. At present the main problem is the instability of the terminal oxidase. Attempts are being made to solve these problems by immobilizing the protein or substituting P-450 by other hemoproteins or iron porphyrin derivatives.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Animals , Hemeproteins/metabolism , Hemin/metabolism , Humans , Hydroxylation , NAD/metabolism , NADP/metabolism , Oxidoreductases/metabolism , Peroxides/metabolismSubject(s)
Fishes , Hemoglobins , Alkanes , Animals , Chemical Phenomena , Chemistry , Circular Dichroism , Cyanides , Ligands , Spectrum AnalysisABSTRACT
In alkaline aqueous solution, the sedimentation coefficients of DeoxyHb, HbO(2) and HbCO of Lampetra fluviatilis L. are 1.9 +/- 0.1 S. This value corresponds to a molecular weight of about 17,000, i.e. the value of a single haem polypeptide chain. In weak acidic conditions, both the non-liganded DeoxyHb and the liganded forms, HbO2 and HbCO, associate to form dimers and oligomers. The monomer-oligomer transitions of these compounds take place at different pH values: DeoxyHb pHO.5 approximately 6.7; HbO(2) and HbCO pH(O.5) approximately 5.9. With respect to the association modus, the equilibrium: 4 Hb &rlhar2; 2 Hb(2)&rlhar2;Hb(4) may be preferred.
ABSTRACT
The kinetics of methaemoglobin substitution reactions have been investigated by the stopped-flow technique. Biphasic reactions have been observed with adult and fetal methaemoglobin. This behaviour is attributed to different reactivities of the chain types. The investigations give evidence of an increasing chain reactivity in the sequence alpha-chain < beta-chain < gamma-chain. The differences increase with the size of the substituting ligand, indicating steric effects.
ABSTRACT
In general, saturation curves for ligand binding by proteins are described by the Adair-equation. The approach to ligand binding with methods of statistical mechanics leads not only to expressions for the Adair-constants, but gives also the possibility of describing the effect of protein-protein interactions on the binding of ligands by proteins. Under the assumption of the superposition-approximation for the potential of mean force between proteins in a solution, which contains also ligand molecules, the variations of the second and third virial coefficient with ligand activity are calculated for several simple model-pontentials. Finally the pair potential of hemoglobin molecules known from X-ray measurements will be approximated by such a simple square-well potential. With that the effect of hemoglobin density on oxygenation will be estimated. One finds for our model system that at relatively high protein density a further increase in density should be accompanied by a decrease in affinity (hindrance of saturation) and an increasing steepness of the slope of the saturation curve.
Subject(s)
Proteins , Calorimetry , Hemoglobins , Kinetics , Ligands , Mathematics , Oxyhemoglobins , Protein Binding , X-Ray DiffractionABSTRACT
The interaction between methemoglobin (MetHb) and macroporous matrices on the basis of polymethacrylates was investigated by means of optical and e.p.r. spectroscopy. The spectroscopic data show that the adsorption of MetHb to imidazole-containing matrices occurs by complex formation between matrix-bound imidazole and the iron of the prosthetic group, with all 4 polypeptide chains of the MetHb molecule being included in the interaction. The adsorption to hydrophobic side chains containing matrices leads, via the protein-matrix interaction, to considerable disturbances of iron protoporphyrin IX in equilibrium or formed from protein-contacts, which are of general importance with respect to the functional variablity and control, respectively, of iron porphyrins in hemoproteins. In case of matrix containing n-hexyl groups deoxyHb is oxidized by O2 to MetHb, instead of being oxygenated to HbO2. Not all prosthetic groups are able to bind N-3. With the increase in hydrophobicity of the matrix a conformational change is enforced leading in the beta-chains to the direct interaction between iron and sulfur of cysteine (beta-cys 92), as it is proved in all cytochrome P-450 and other model compounds.
Subject(s)
Methemoglobin , Polyhydroxyethyl Methacrylate , Polymethacrylic Acids , Animals , Cattle , Chemical Phenomena , Chemistry , Electron Spin Resonance Spectroscopy , Methemoglobin/analogs & derivatives , Spectrophotometry , Spectrophotometry, UltravioletABSTRACT
The activity of urease in microcapsules formed by a symplex membrane from cellulose sulphate and polydimethyldiallyl ammonium chloride is studied and compared with that of free urease in solution. Both free and encapsulated urease exhibits a similar pH optimum. The interaction of urease with cellulose sulphate causes a decrease of the activity down to 35 to 38%. In spite of its lower absolute activity the encapsulated urease shows a higher long time stability than the free urease. The presented immobilization method for urease could be of importance for an active detoxification in uremia.
Subject(s)
Enzymes, Immobilized/administration & dosage , Polyethylenes , Quaternary Ammonium Compounds , Urease/administration & dosage , Capsules , Cellulose/analogs & derivatives , Enzymes, Immobilized/metabolism , Hydrogen-Ion Concentration , Kinetics , Polymers , Urease/metabolismABSTRACT
1. Six different hemoglobin (Hb) fractions were isolated and characterized from the larvae of Chironomus thummi thummi using column chromatographic procedures. 2. Chromatographic and sedimentation-analytic studies (sedimentation coefficients of 2.0 +/- 0.2 (S)) have shown three Hb fractions to exist basically in a monomeric form. The molecular weight of component M-2 was determined by sedimentation equilibrium technique to be 15,470 +/- 400. The dimeric Hb was found to have sedimentation coefficients of 3.0 +/- 0.1 (S) in the weakly acidic pH region. In alkaline milieu, the reversible dissociation proceeds into the monomeric molecules (S20, W = 1.9 +/- 0.1 (S)). Molecular weights vary between pH 5.7 and 9.8 not only with hydrogen ion concentration, but also with protein concentration in correspondence with a dissociation-association equilibrium consisting of monomers and dimers. 3. For the Hb fraction M-2, a friction ratio of f/fo = 1.03 was calculated, suggesting an almost spherical shape of this protein. In contrast, the dimeric component appears to have a much more asymmetric structure (f/fo = 1.19). 4. The indivdual MetHb fractions bind the ligands: fluoride, imidazole and azide with different affinities.