Bovine Erythrocyte Superoxide Dismutase Research Articles

Bovine Erythrocyte Superoxide Dismutase: ISOLATION AND CHARACTERIZATION OF TRYPTIC, CYANOGEN BROMIDE, AND MALEYLATED TRYPTIC PEPTIDES

Tryptic and cyanogen bromide peptides were isolated from reduced and S-carboxymethylated bovine erythrocyte superoxide dismutase, and maleylated tryptic peptides were isolated from the individual cyanogen bromide fragments. Based upon end group analyses of the intact protein and the yields and partial sequence analyses of these peptides, a partial sequence of the enzyme was deduced. From these data it was concluded that the 2 subunits of bovine dismutase appear to be chemically identical. Each subunit is acetylated at the NH2 terminus, has lysine at the COOH terminus, and contains no tryptophan.

Preliminary Report on the Electron Paramagnetic Resonance Spectra of Single Crystals of Bovine Erythrocyte Superoxide Dismutase

Abstract Electron paramagnetic resonance spectra were recorded from single monoclinic crystals of bovine erythrocyte superoxide dismutase. Spectra were taken at various angles of rotation about the c (or blue optic) axis (Richardson, D. C., Bier, C. J., and Richardson, J. S. (1972), J. Biol. Chem. 247, 6368–6369) and about the a or b (green optic) axis of the crystal. Rotation about the blue axis (H in ab plane) yields the information that the Z components of all Cu(II) g tensors lie substantially in the ab plane, and this is confirmed by rotation about one of the green axes which yielded only very small Z component resonances (H in ac or bc plane). A maximum of eight hyperfine components were observed regardless of the orientation of the crystal. There appear to be two distinct groups of eight symmetrically degenerate (at least along their Z axes) Cu(II) ions, and the common Z axes of these two groups are off parallel by only a few degrees.

Observations on the oxidation-reduction properties of bovine erythrocyte superoxide dismutase.

Observations on the oxidation-reduction properties of bovine erythrocyte superoxide dismutase.

On the Stability of Bovine Superoxide Dismutase: THE EFFECTS OF METALS

Abstract 1. Apo-superoxide dismutase was more labile toward a variety of inactivating stresses than was the holoenzyme. 2. Cu++ restored catalytic activity to the apoenzyme and markedly enhanced its thermal stability but Cu++ plus Zn++ were needed to attain the stability of the native enzyme. 3. Co++ or Hg++ were able to replace Zn++ in increasing the thermal stability of the Cu++-repleted apoenzyme. Indeed, the enzyme containing Hg++ + Cu++ was more stable than the native superoxide dismutase. In contrast no metal was found which could replace Cu++ in restoring activity to the apoenzyme. 4. Arrhenius plots showed that rates of irreversible inactivation were a biphasic function of temperature in the case of apo-superoxide dismutase but were monophasic in the presence of those metals which affected thermal stability. Metal binding apparently changed the pathway of thermal inactivation. 5. Since excess Cu++ could not substitute for Cu++ + Zn++ and since excess Zn++ could not prevent the effect of Cu++ we conclude that the Cu++ site cannot bind Zn++ and the Zn++ site cannot bind Cu++, although it can bind Co++ or Hg++. 6. Native superoxide dismutase retained much of its activity in the presence of 10.0 m urea or 4% sodium dodecyl sulfate and, although high concentrations of guanidinium chloride did inhibit, its effects were reversible by dialysis or dilution. 7. Guanidinium-chloride did however perturb the structure of the holoenzyme in a way which rendered its metals accessible to EDTA such that exposure to EDTA plus guanidinium chloride resulted in progressive inactivation. 8. It appears likely, on the basis of data available at present, that Zn++ plays a structural role in the bovine erythrocyte superoxide dismutase and lends it enhanced stability whereas Cu++ is directly involved in the catalytic cycle.

Studies on the reconstitution of bovine erythrocyte superoxide dismutase: III. Evidence for a strong interdependence between Cu 2+ and Zn 2+ binding in the expression of the spectroscopic properties of the native protein and for a close proximity of the Zn 2+ and Cu 2+ sites

1. 1. Conditions are described for reconstitution of apo superoxide dismutase to native protein in high yield with no side products. 2. 2. 67 Zn (I = 5 2 ) has been substituted in place of normal zinc with no observable broadening of the EPR spectrum. 3. 3. Excess cyanide added to the 63Cu-substituted protein yields a low-field hyperfine line having a superhyperfine pattern clearly arising from an interaction with three nitrogen nuclei. 4. 4. Cu 2+ may bind so as to form a Zn 2+-binding site and Zn 2+ may bind forming a native Cu 2+-binding site. 5. 5. When present in excess, Cu 2+ bind in close proximity to one another, and an EPR spectrum characteristic of a magnetic dipolar coupling between at least two Cu 2+ has been observed.

Studies on the reconstitution of bovine erythrocyte superoxide dismutase: II. Some observations on the nature of catalyzed superoxide anion dismutation as an enzymatic activity

1. 1. A spurious form of superoxide dismutase activity has been found to result from the simple addition of Cu 2+ to aqueous solutions of apo dismutase. Cu 2+ are bound to the protein in such a manner as to possess no spectral property in common with copper bound in native form, and this activity can be partially inhibited by EDTA. 2. 2. Conditions have been found under which reconstitution of superoxide dismutase activity requires stoichiometric quantities of both Cu 2+ and Zn 2+. 3. 3. The six sulfur atoms of bovine dismutase are shown to exist as two sulfhydryl and two disulfide groups in the apo protein. These are available for titration with mercurials only under denaturing contions. 4. 4. Preliminary evidence is presented which indicates sulfhydryl groups to be essential in reforming native protein.

Studies on the reconstitution of bovine erythrocyte superoxide dismutase: I. The presence of four divalent metal-binding sites on the apo protein which are different from the native sites

1. 1. The extinction coefficient ( E 1 cm 1 % ) of metal-free protein at 258 nm is 0.85±10% based on Lowry determinations of solutions of bovine serum albumin. 2. 2. The two tyrosine residues of superoxide dismutase fluorescence at λ max = 320 nm when excited at λ max = 278 nm , and the empirical intensity of the emission is the same in both apo and native proteins. 3. 3. The binding of Cu 2+, Zn 2+ and Hg 2+ to apo protein has been studied by fluorescence, polarographic, and optical absorption methods. 4. 4. The apo protein may bind four each of these ions to independent sites which differ in an unknown manner from those of the native protein.

Nuclear magnetic relaxation dispersion in protein solutions: V. Bovine erythrocyte superoxide dismutase

The paramagnetic contribution R p to the spin-lattice relaxation rate of solvent protons in aqueous solutions of bovine erythrocyte superoxide dismutase has been measured for magnetic fields in the range 5 Oe to 12 kOe and temperatures in the range 0 °C to 25 °C. We find a peak in R p as a function of magnetic field, as has been observed recently by several investigators for Mn 2+-macromolecular complexes, indicating that the spin-lattice relaxation time τ s of the Cu 2+ electron spin moments is magnetic field dependent. τ s is approx. 5·10 −10 s at low field and begins to increase above approx. 750 Oe. From the magnitude of R p and its qualitative behavior with magnetic field and temperature, combined with previous EPR and inhibition studies, we conclude that there is at least one rapidly exchanging water molecule (exchange time τ M in the range 4·10 −6 to 10 −8 s) on each of the two Cu 2+ per protein molecule. We speculate that the water binding sites on the Cu 2+, with a Cu—O bond distance approx. 2.0 Å, are similarly accessible to the superoxide anion and are the loci for the catalytic activity of the enzyme.

Anion Binding to Bovine Erythrocyte Superoxide Dismutase: EVIDENCE FOR MULTIPLE BINDING SITES WITH QUALITATIVELY DIFFERENT PROPERTIES

Abstract The binding of azide, thiocyanate, cyanate, and cyanide to bovine erythrocyte superoxide dismutase has been studied by means of optical spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and nuclear magnetic relaxation dispersion (NMRD). The results show that there are at least four anion binding sites on the protein, and these are divided equally into two classes. One of these classes of binding sites strongly binds 2 moles of azide producing an altered EPR spectrum, no change in the NMRD, and only a minor change in the visible optical spectrum. These two binding sites are considered to be the Zn2+ ions of the protein. The other class of binding sites binds 2 moles of cyanide, lowering the NMRD to the diamagnetic level, and producing substantial changes in the optical and EPR spectra. These two binding sites are considered to be the 2 Cu2+ ions of the protein. Thiocyanate and cyanate appear to bind only to the Zn2+, having no effect on the NMRD and minor effects on the optical spectrum, although each ion produces a characteristic EPR spectrum. Addition of excess azide to the protein results in the appearance of an intense charge transfer band near 373 nm, produces minor changes in the 2-azide complex EPR spectrum and lowers the NMRD to the diamagnetic level. The presence of excess cyanide produces an absorption band at 530 nm and a unique EPR spectrum (Rotilio, G., Finazzi Agro, A., Calabrese, L., Bossa, F., Guerrieri, P., and Mandovi, B. (1971) Biochemistry, 10, 611). The data have been rationalized in terms of a model in which zinc and copper are relatively close to each other in the protein, perhaps existing in a ligand-bridged bimetal complex.

Substrate-induced chemiluminescence of xanthine oxidase and aldehyde oxidase

Milk xanthine oxidase and rabbit liver aldehyde oxidase produce chemiluminescence when operating on their substrates. This luminescence can be measured by a Beckman scintillation counter with the coincidence circuit turned off. Hydrogen peroxide markedly enhances the luminescence, and catalase inhibits the light signal by more than 95%. The inhibition of luminescence by sulfite, cysteine, and bovine erythrocyte superoxide dismutase indicates that a free radical, probably the superoxide ion (O 2⨪), is responsible for the light signal.