Bo G. Malmström

The Specific Binding Of Iron(Iii) And Copper(Ii) To Transferrin And Conalbumin

Abstract The binding of Fe3+ to transferrin has been studied by pH titrations and equilibrium dialysis. In the pH range studied (7.5–8.9), 3 H+ are released for each Fe3+ bound. In the dialysis experiments, EDTA and citrate were used as competing chelating agents. It was found that the binding of Fe3+ to transferrin may be described as coordination to two equivalent and independent sites. The nature of the binding sites in transferrin as well as conalbumin has been studied by measurements of ESR and visible spectra. The ESR spectra of the Fe complexes indicate that the metal is present as Fe3+. Measurements of ESR and optical absorption as a function of the degree of binding in transferrin, coupled with the known binding constants, exclude strong interactions between the 2 Fe3+, and indicate that the distance between them is greater than 9 A. Attempts have been made to fit the experimental data to a spin-Hamiltonian and the results discussed in terms of the geometrical configuration of the complex. In the case of both Fe3+ and Cu2+, the ESR spectra of transferrin and conalbumin are very similar. The Cu2+ spectra show hyperfine structure from 2–3 N nuclei. Urea denaturation destroys the specific bonding of Fe3+ in transferrin, as shown by the parallel loss of color and ESR signal. The nature of the chelating sites in transferrin and conalbumin is discussed in relation to the present and earlier data. It is suggested that each chelating site involves 2 imidazole groups of the protein in addition to 3 tyrosyl residues.

Electron spin resonance of copper proteins and some model complexes

Electron spin resonance spectra of a number of copper proteins (laccase, cerulo-plasmin, erythrocuprein and Cu2+-carboxypeptidase) and of some small-molecule complexes, with known coordinating atoms, have been recorded, mainly in frozen aqueous solutions. The method used in evaluating the spectra has been discussed, and the g-values and the hyperfine structure constants as well as the absorption maxima in the visible spectra have been determined. A qualitative theory for correlating the experimental data with the bonding of the Cu(II) ion has been given. An observed trend in g-values and hyperfine structure constants suggests that the metal is coordinated to N in the proteins. The metal in the oxidative enzymes (laccase, ceruloplasmin) appears coordinated in a unique manner, as evidenced by their exceptionally low hyperfine structure constants. This can be related to a high degree of derealization of the unpaired electron. The unique bonding disappears on denaturation, and its possible relation to the oxidase activity of the proteins has been discussed.

Quantitative electron spin resonance studies on native and denatured ceruloplasmin and laccase.

The effect of urea denaturation on the visible absorption, electron spin resonance spectrum and oxidase activity of ceruloplasmin has been investigated. Some measurements of the effect on optical rotation and ultraviolet absorption have also been performed. The denaturation destroys the specific bonding of Cu 2+ , characteristic for native ceruloplasmin as evidenced by electron spin resonance spectra. The loss of color and oxidase activity follows the same time-course and concentration dependence as the change in bonding, while comparable changes in optical rotation and ultraviolet difference spectra require more drastic denaturation. These findings indicate that the strong visible absorption is related to the specific bonding of Cu 2+ , and that this bonding is necessary for catalytic activity. With both ceruloplasmin and laccase, the Cu 2+ signal of the native protein only corresponds to about 50% of the total copper content, as determined both by chemical analysis and by measurements of the electron spin resonance signal intensities after release and oxidation of protein-bound copper with perchloric acid. The possibility that the specific bonding may involve an interaction between Cu 2+ and Cu 1+ is discussed.

The role of copper in the catalytic action of laccase and ceruloplasmin

Abstract 1. 1. The rate of reoxidation of ascorbic acid-reduced copper in laccase (EC 1.10.3.2) and ceruloplasmin has been measured spectrophotometrically in a stopped-flow apparatus. The kinetics of reduction of Cu 2+ by various substrates has been studied by electron-spin resonance absorption. The rate of formation and decay of a free radical formed with p -phenylenediamine as substrate has also been measured, and the radical has been identified as the positive ion. 2. 2. The rate of O 2 consumption, calculated from the kinetic parameters on the basis of a mechanism involving reduction of Cu 2+ by the substrate followed by reoxidation by O 2 , agrees well with the experimentally determined rate, thus providing strong evidence for the correctness of this mechanism. 3. 3. In a few cases, the experimental curves for Cu 2+ and free radical have been compared with calculated curves, obtained by numerical integrations of kinetic equations with a digital computer. The results indicate that the radical decay occurs by a reaction not involving the enzymes. 4. 4. Electron-spin resonance measurements of a bacterial copper protein, azurin, indicate that small hyperfine splittings, as observed with laccase and ceruloplasmin, may occur without an exchange interaction between Cu 2+ and Cu 1+ . It is suggested that the Cu 1+ of laccase and ceruloplasmin may bind the substrates by interacting with their π-electrons.

An electron-spin-resonance study of the interaction of manganous ions with enolase and its substrate.

The interaction of Mn++ with enolase and with DL-2-phosphoglyceric acid has been studied with the electron-spin-resonance technique. In both cases the hyperfine structure disappears in the complex, and this makes it possible to determine the dissociation constants. The results obtained by the electron-spn-resonance studies agree with those obtained from chemical equilibrium measurements. The binding studies were carried out at a high ionic strength (μ = 0.50) which was found to suppress non-specific binding to the enzyme. A comparison with the activation kinetics shows that activation involves the binding of a single ion of Mn++ per mole of enzyme. The data also indicate that the metal ion is involved in binding the substrate to the enzyme.

The purification of yeast enolase by zone electrophoresis and ion-exchange chromatography, and the existence of several active forms of the enzyme.

Abstract 1. 1. A new method for the purification of yeast enolase has been described. The procedure involves zone electrophoresis in cellulose columns, and specific adsorption of the enzyme on the Mg form of a cation exchanger, sulfomethylcellulose. In a typical preparation, 1.7 g. of pure enolase was obtained from 3 kg. of dried yeast. 2. 2. The purified enzyme shows a high degree of homogeneity in zone electrophoresis and in the ultracentrifuge. Its specific activity is the same as that of the enzyme prepared by the method of Warburg and Christian (1). 3. 3. It has been shown that the crystallized enzyme can contain several electrophoretic components, all having the same specific activity and sedimentation constant. 4. 4. Some advantages of the new purification procedure and the possible origin of the different forms of the enzyme have been discussed.

The metal-ion activation of 3-phosphoglycerate kinase in correlation with metal-binding studies

Abstract The binding of Mn ++ to 3-phosphoglycerate kinase, and to ADP and ATP, has been studied at pH 7.1 and 38 °. The influence of the concentrations of Mn ++ and of the various substrates on the rate of the enzymic reaction has also been investigated at the same conditions. The protein displays only a weak, nonspecific interaction with Mn ++ . By correlating the kinetic and binding data, it is possible to show that activation does not involve binding of Mn ++ to the enzyme but rather the formation of a Mn ++ -ATP complex. The significance of the results in relation to the general problem of the mechanism of metal-ion activation of enzymes is discussed.

The degradation of yeast enolase with leucine aminopeptidase and carboxypeptidase without change in enzymic activity; N- and C-terminal residues of the enzyme.

Abstract 1. 1. The earlier finding 5 that enolase consists of a single chain having alanine as the N-terminal amino acid has been confirmed by the phenylisothiocyanate method. The only C-terminal amino acid in the enzyme is leucine, as shown by the carboxy-peptidase method. Enolase a and b do not differ with respect to N- or C-terminal groups. 2. 2. It has been found that the enolase molecule can be extensively degraded with leucine aminopeptidase or carboxypeptidase without loss of activity. Not only the velocity but also the extent of digestion achieved is dependent on the molar ratio between peptidase and enolase. Maximally, about 150 residues can be removed from either end. 3. 3. Some implications of the results for the study of the structure of enzymes are discussed.