Multisite fluorescence in proteins with multiple tryptophan residues. Apomyoglobin natural variants and site-directed mutants

Time-resolved fluorescence experiments were carried out on a variety of apomyoglobins with one or two tryptophan (Trp) residues located at invariant positions 7 and 14 in the primary sequence. In all cases, the Trp fluorescence kinetics were resolved adequately into two discrete lifetime domains, and decay-associated spectra (DAS) were obtained for each decay component. The DAS resolved for unfolded proteins were indistinguishable by position of the emission maxima and the spectral shapes. The folded proteins revealed noticeable differences in the DAS, which relate to the diverse local environments around the Trp residues in the individual proteins. Furthermore, the DAS of wild-type protein possessing two Trp residues were simulated well by that of one Trp mutants either in the native, molten globule, or unfolded states. Overall, employing Trp fluorescence and site-directed mutagenesis allowed us to highlight the conformational changes induced by the single amino acid replacement and generate novel structural information on equilibrium folding intermediates. Specifically, it was found that conformational fluctuations in the local cluster around the evolutionarily conserved Trp(14) are very similar in the native and molten globule states of apomyoglobins. This result indicates that residues in the E and B helices contributing to this cluster are most likely involved in the stabilization of the overall architecture of the structured molten globule intermediate.     

A calcium-specific conformational response of parvalbumin

The single tryptophan containing isotype III parvalbumin from codfish (Gadus callarius) was purified by a modified procedure and was shown to be homogeneous by a number of biochemical techniques. Sequence analysis established the location of the single tryptophan in position 102 of the 108 amino acid primary sequence. Atomic absorption spectroscopy showed that trichloroacetic acid (TCA) precipitation was more effective in parvalbumin decalcification compared to the more commonly used method of EGTA treatment. Magnesium induced steady-state fluorescence spectral changes of the EGTA-treated, but not the TCA-treated, parvalbumin. Steady-state fluorescence and circular dichroism spectra showed that calcium, but not magnesium, induced a conformational response in the TCA-treated protein. The fluorescence decay of the calcium-loaded native (holo) cod III parvalbumin was best described by two decay time components. By contrast, three lifetime components were necessary to describe the fluorescence decay of the metal-free (apo) protein. The decay-associated spectra of each temporal component were obtained. Collectively, these results demonstrate that it is possible for a parvalbumin to display a calcium-specific response.     

Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase

A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.     

Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 1. Theory and applications

Individual fluorescence spectra for species in a heterogeneous system can be determined by using differences between the rotational correlation times of those components. Each spectrum derived is associated with a particular fluorescence anisotropy decay function; hence, they are anisotropy decay associated spectra (ADAS). We have previously shown [Knutson, J. R., Walbridge, D. G., & Brand, L. (1982) Biochemistry 21, 4671-4679] that a system containing different decay functions for total intensity can be resolved into constituent decay-associated spectra. ADAS extends the technique into the realm of fluorescence polarization, making use of the often disparate Brownian rotations found in heterogeneous biochemical systems. In this paper, we present the basic theory for ADAS in various heterogeneous systems and then present an example of ADAS resolving a binary mixture of macromolecules into "fast-rotor" (smaller or more mobile) and "slow-rotor" (larger or less mobile) components. They correctly superimpose spectra taken for the unmixed components. In the companion paper [Davenport, L., Knutson, J. R., & Brand, L. (1986) Biochemistry (following paper in this issue)], a specific application to a problem of importance of lipid biochemistry--e.g., the origin of the membrane probe order parameter in lipid bilayers--is presented, demonstrating the role rotational heterogeneity may play in biochemical fluorescence.     

Time-resolved spectral observations of cadmium-enriched cadmium sulfide nanoparticles and the effects of DNA oligomer binding

We measured the steady-state and time-resolved fluorescence spectral properties of cadmium-enriched nanoparticles (CdS-Cd2+). These particles displayed two emission maxima, at 460 and 580 nm. The emission spectra were independent of excitation wavelength. Surprisingly, the intensity decays were strongly dependent on the observation wavelength, with longer decay times being observed at longer wavelengths. The mean lifetime increased from 150 to 370 ns as the emission wavelength was increased from 460 to 650 nm. The wavelength-dependent lifetimes were used to construct the time-resolved emission spectra, which showed a growth of the long-wavelength emission at longer times, and decay-associated spectra, which showed the longer wavelength emission associated with the longer decay time. These nanoparticles displayed anisotropy values as high as 0.35, depending on the excitation and emission wavelengths. Such high anisotropies are unexpected for presumably spherical nanoparticles. The anisotropy decayed with two correlation times near 5 and 370 ns, with the larger value probably due to overall rotational diffusion of the nanoparticles. Addition of a 32-base pair oligomer selectively quenched the 460-nm emission, with less quenching being observed at longer wavelengths. The time-resolved intensity decays were minimally affected by the DNA, suggesting a static quenching mechanism. The wavelength-selected quenching shown by the nanoparticles may make them useful for DNA analysis.     

Study of the time-resolved tryptophan fluorescence of crystalline alpha-chymotrypsin

The tryptophan environments in crystalline alpha-chymotrypsin were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was resolved by time-correlated fluorescence spectroscopy. The fluorescence decays at 296-nm laser excitation and various emission wavelengths could be characterized by a triple-exponential function with decay times tau 1 = 150 +/- 50 ps, tau 2 = 1.45 +/- 0.25 ns, and tau 3 = 4.2 +/- 0.4 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 325, 332, and 343 nm. The three decay components in this enzyme can be correlated with X-ray crystallographic data [Birktoft, J.J., & Blow, D.M. (1972) J. Mol. Biol. 68, 187-240]. Inter- and intramolecular tryptophan-tryptophan energy-transfer efficiencies in crystalline alpha-chymotrypsin were computed from the accurately known positions and orientations of all tryptophan residues. These calculations indicate that the three fluorescence decay components in crystalline alpha-chymotrypsin can be assigned to three distinct classes of tryptophyl residues. Because of the different proximity of tryptophan residues to neighboring internal quenching groups, the decay times of the three classes are different. Decay tau 1 can be assigned to Trp-172 and Trp-215 and tau 2 to Trp-51 and Trp-237, while the tryptophyl residues 27, 29, 141, and 207 all have decay time tau 3.     

Solution conformation of EcoRI restriction endonuclease changes upon binding of cognate DNA and Mg2+ cofactor

EcoRI endonuclease has two tryptophans at positions 104 and 246 on the protein surface. A single tryptophan mutant containing Trp246 and a single cysteine labeling site at the N-terminus was used to determine the position of the N-terminus in the protein structure. The N-termini of EcoRI endonuclease are essential for tight binding and catalysis yet are not resolved in any of the crystal structures. Resonance energy transfer was used to measure the distance from Trp246 donor to IAEDANS or MIANS acceptors at Cys3. The distance is 36 A in apoenzyme, decreasing to 26 A in the DNA complex. Molecular modeling suggests that the N-termini are located at the dimer interface formed by the loops comprising residues 221-232. Protein conformational changes upon binding of cognate DNA and cofactor Mg(2+) were monitored by tryptophan fluorescence of the single tryptophan mutant and wild-type endonuclease. The fluorescence decay of Trp246 is a triple exponential with lifetimes of 7, 3.5, and 0.7 ns. The decay-associated spectra of the 7- and 3.5-ns components have emission maxima at approximately 345 and approximately 338 nm in apoenzyme, which shift to approximately 340 and approximately 348 nm in the DNA complex. The fluorescence quantum yield of the single tryptophan mutant drops 30% in the DNA complex, as compared to 10% for wild-type endonuclease. Fluorescence changes of Trp104 upon binding of DNA were inferred by comparison of the decay-associated spectra of wild type and single tryptophan mutant. Fluorescence changes are related to changes in proximity and orientation of quenching functional groups in the tryptophan microenvironments, as seen in the crystal structures.     

Mechanism of the down regulation of photosynthesis by blue light in the Cyanobacterium synechocystis sp. PCC 6803

Exposure to blue light has previously been shown to induce the reversible quenching of fluorescence in cyanobacteria, indicative of a photoprotective mechanism responsible for the down regulation of photosynthesis. We have investigated the molecular mechanism behind fluorescence quenching by characterizing changes in excitation energy transfer through the phycobilin pigments of the phycobilisome to chlorophyll with steady-state and time-resolved fluorescence excitation and emission spectroscopy. Quenching was investigated in both a photosystem II-less mutant, and DCMU-poisoned wild-type Synechocystis sp. PCC 6803. The action spectra for blue-light-induced quenching was identical in both cell types and was dominated by a band in the blue region, peaking at 480 nm. Fluorescence quenching and its dark recovery was inhibited by the protein cross-linking agent glutaraldehyde, which could maintain cells in either the quenched or the unquenched state. We found that high phosphate concentrations that inhibit phycobilisome mobility and the regulation of energy transfer by the light-state transition did not affect blue-light-induced fluorescence quenching. Both room temperature and 77 K fluorescence emission spectra revealed that fluorescence quenching was associated with phycobilin emission. Quenching was characterized by a decrease in the emission of allophycocyanin and long wavelength phycobilisome terminal emitters relative to that of phycocyanin. A global analysis of the room-temperature fluorescence decay kinetics revealed that phycocyanin and photosystem I decay components were unaffected by quenching, whereas the decay components originating from allophycocyanin and phycobilisome terminal emitters were altered. Our data support a regulatory mechanism involving a protein conformational change and/or change in protein-protein interaction which quenches excitation energy at the core of the phycobilisome.     

Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29. Effects of zeaxanthin, pH and phosphorylation.

Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll A fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.     

Modification of energy-transfer processes in the cyanobacterium, Arthrospira platensis, to adapt to light conditions, probed by time-resolved fluorescence spectroscopy

In cyanobacteria, the interactions among pigment–protein complexes are modified in response to changes in light conditions. In the present study, we analyzed excitation energy transfer from the phycobilisome and photosystem II to photosystem I in the cyanobacterium Arthrospira (Spirulina) platensis. The cells were grown under lights with different spectral profiles and under different light intensities, and the energy-transfer characteristics were evaluated using steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopy techniques. The fluorescence rise and decay curves were analyzed by global analysis to obtain fluorescence decay-associated spectra. The direct energy transfer from the phycobilisome to photosystem I and energy transfer from photosystem II to photosystem I were modified depending on the light quality, light quantity, and cultivation period. However, the total amount of energy transferred to photosystem I remained constant under the different growth conditions. We discuss the differences in energy-transfer processes under different cultivation and light conditions.     

A new method for decay-associated fluorescence spectroscopy. Application to the tryptophan zwitterion

We describe a new method for decay associated fluorescence spectroscopy using synchrotron radiation as the excitation pulse and a photon counting technique. This method is based on the determination of the difference between the barycenters of the exciting pulse and of the fluorescence response at several wavelengths. It is applicable to the case where individual decay times are independent of emission wavelength. Coupled to the analysis of the decay curve at only one emission wavelength, this method reduces the time devoted to the numerical analysis and avoids the spectral distortion due to the lamp profile. The results obtained by this method on indole, the tryptophan zwitterion, and N-acetyl-tryptophan are presented. Results are compared to those obtained by two other methods: Determination of the fluorescence decay parameters by deconvolution analysis at several emission wavelengths. Photon counting of the fluorescence spectrum emitted during a selected time window after the excitation pulse.     

Fluorescence decay and spectral evolution in intact photosystem I of higher plants

A photosystem I preparation from maize, containing its full antenna complement (PSI-200) and in which detergent effects on chlorophyll coupling are almost completely absent, has been studied by time-resolved fluorescence techniques with approximately 5 ps resolution at 280 and 170 K in the wavelength interval of 690-780 nm. The data have been analyzed in terms of both the decay-associated spectra (DAS) and the time-resolved emission spectra (TRES). As in a previous room temperature study [Turconi, S., Weber, N., Schweitzer, D., Strotmann, H., and Holzwarth, A. R. (1994) Biochim. Biophys. Acta 1187, 324-334], the 280 K decay is well described by three DAS components in the 11-130 ps time range, the fastest of which displays both positive and negative amplitudes characteristic of excitation transfer from the bulk to the red antenna forms. Both the 57 and 130 ps components have all positive amplitudes and describe complex decay and equilibration processes involving the red forms. At 170 K, four major components in the 10-715 ps time range are required to describe the decay. The fastest represents bulk to red form transfer processes, while the 55, 216, and 715 ps decays, with all positive amplitudes, have maxima near 720, 730, and 740 nm, respectively, in accord with previous steady-state fluorescence measurements. The width and asymmetry of these DAS indicate that they are spectrally complex and represent decay and equilibration processes involving the red forms. Spectral evolution during the fluorescence decay process was analyzed in terms of the TRES. The red shifting of the TRES was analyzed in terms of the first central spectral moment (mean spectral energy) which is biexponential at both temperatures. The slower component, which describes equilibration between the red forms, leads to spectral red shifting during the entire fluorescence decay process, and the mean lifetimes of the spectral moments at 280 and 170 K (86 and 291 ps, respectively) are similar to the mean lifetimes of the fluorescence decays (119 and 384 ps, respectively). Thus, both spectral evolution and the trapping-associated fluorescence decay occur on a similar time scale, and both processes display a very similar temperature sensitivity. On the basis of these data, it is concluded that trapping in PSI-200 is to a large extent rate-limited by excitation diffusion in the antenna and in particular by the slow "uphill" transfer from the low-energy forms to the bulk and/or inner core chlorophyll molecules.     

Time-resolved fluorescence spectroscopy of human adenosine deaminase: effects of enzyme inhibitors on protein conformation

Adenosine deaminase, a purine salvage enzyme essential for immune competence, was studied by time-resolved fluorescence spectroscopy. The heterogeneous emission from this four-tryptophan protein was separated into three lifetime components: tau 1 = 1 ns and tau 2 = 2.2 ns an emission maximum at about 330 nm and tau 3 = 6.3 ns with emission maximum at about 340 nm. Solvent accessibility of the tryptophan emission was probed with polar and nonpolar fluorescence quenchers. Acrylamide, iodide, and trichloroethanol quenched emission from all three components. Acrylamide quenching caused a blue shift in the decay-associated spectrum of component 3. The ground-state analogue enzyme inhibitor purine riboside quenched emission associated with component 2 whereas the transition-state analogue inhibitor deoxycoformycin quenched emission from both components 2 and 3. The quenching due to inhibitor binding had no effect on the lifetimes or emission maxima of the decay-associated spectra. These observations can be explained by a simple model of four tryptophan environments. Quenching studies of the enzyme-inhibitor complexes indicate that adenosine deaminase undergoes different protein conformation changes upon binding of ground- and transition-state analogue inhibitors. The results are consistent with localized structural alterations in the enzyme.     

Global analysis of steady-state polarized fluorescence spectra using trilinear curve resolution.

Global analysis using trilinear curve resolution is described and shown to be a powerful method for the resolution of polarized fluorescence data arrays, in which the measured fluorescence intensity is a separable function of polarization orientation, excitation wavelength, and emission wavelength. This methodology is applicable to mixtures the components of which have linearly independent excitation and emission spectra and distinct anisotropies. Normalized excitation and emission spectra of individual components can be uniquely determined without prior assumptions concerning spectral shapes (e.g., sum of Gaussians) and without the uncertainties inherent in bilinear techniques such as principal component analysis or factor analysis. The normalized excitation and emission vectors are combined with the total absorption spectrum of the multicomponent mixture to compute absolute absorption and emission spectra. The precision of this methodology is evaluated as a function of noise, overlap, relative intensity, and anisotropy difference between components using simulated mixtures of the DNA bases. The ability of this method to extract individual spectra from steady-state fluorescence data arrays is illustrated for mixtures containing two and three components.     

Tryptophan fluorescence of terminal deoxynucleotidyl transferase: effects of quenchers on time-resolved emission spectra

Terminal deoxynucleotidyl transferase (EC 2.7.7.31) is a eucaryotic DNA polymerase that does not require a template. The tryptophan environments in calf thymus terminal transferase were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was separated by time-resolved emission spectroscopy. Nanosecond fluorescence decays at 296-nm excitation and various emission wavelengths were deconvolved by global analysis, assuming that the lifetimes but not the relative weighting factors were independent of emission wavelength. The data were fit to three exponentials of lifetimes tau 1 = 1.4 ns, tau 2 = 4.5 ns, and tau 3 = 7.7 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 328, 335, and 345 nm. The accessibility of individual tryptophan environments to polar and nonpolar fluorescence quenchers was examined in steady-state and time-resolved experiments. In the presence of iodide and acrylamide, the steady-state emission spectra shift to the blue. However, at low quencher concentrations, the emission from the 7.7-ns component (maximum 345 nm) is hardly affected, suggesting that this hydrophilic tryptophan environment is buried within the protein. On the other hand, the red shift in the steady-state emission spectrum in the presence of trichloroethanol indicates that the 1.4-ns component (maximum 328 nm) is an exposed hydrophobic tryptophan environment. The results are consistent with an inside-out model for terminal transferase protein, with the more hydrophobic tryptophan(s) near the surface and the most hydrophilic tryptophan(s) in the core.     

去镁叶绿素置换细菌去镁叶绿素对紫细菌反应中心皮秒荧光的影响

选择597 nm作为激发波长,探测范围为600～900 nm的荧光特性,分析了天然反应中心和两种去镁叶绿素置换的紫细菌反应中心的荧光发射光谱.借助细菌叶绿素、细菌去镁叶绿素和植物去镁叶绿素的荧光光谱,对相关组分进行了归类.实验结果表明选择性地置换细菌去镁叶绿素影响了荧光光谱的组成.在天然反应中心、BpheB置换的反应中心和BpheA,B置换的反应中心中可分别解析到4、3和2个荧光发射组分.研究肯定荧光发射组分与去镁叶绿素的结合存在对应关系.实验还分别在686.4、674.1和681.1 nm处测定了不同反应中心内的原初电子供体P的激发态通过荧光衰减的过程,观测到衰减动力学上的差异.说明去镁叶绿素置换影响了细菌反应中心内激发光能传递和原初光化学反应过程.     

Investigation of noncalcium interactions of fura-2 by classical and synchronous fluorescence spectroscopy.

Several authors have reported unexpected intracellular spectra of both indo-1 and fura-2. One of the major methodological problems in the evaluation of calcium concentration using fluorescent probes is that it is assumed that only two forms of the dyes are detectable within the cells. We show in this study of fura-2 properties that this calcium probe is pH-sensitive and able to bind to cellular proteins. The excitation spectra of protonated and protein-bound forms of fura-2 exhibit a maximum in the same region as that associated with the calcium-free form (i.e., near 365 nm). The very small shift in the excitation spectra upon proton or protein binding precludes the use of classical methods to determine the spectral composition of mixtures of several forms of fura-2. We therefore used the synchronous fluorescence technique to detect the protein-bound form of fura-2 selectively, in order to assess the pH dependence of the fura-2/protein interaction. The nonspecific binding of fura-2 to proteins is reinforced at acidic pH and inhibited by calcium. The fact that the same type of interaction was found between fura-2 and poly-L-lysine suggests that it could be mediated by basic amino acids. Because of the strong overlap of the excitation spectrum of the unprotonated free fura-2 with those associated with the protonated and protein-bound forms, a cytoplasmic acidification may lead to an artifactual measurement of low calcium levels.     

Analysis of time-resolved emission spectra of oriented phycobilisomes

The polarized time-resolved (in ps range) fluorescence spectra of phycobilisomes obtained from cyanobacteria Tolypothrix tenuis embedded in poly(vinyl alcohol) films and oriented by film stretching have been analysed. Fluorescence spectra were deconvoluted on Gaussian components supposing the same positions of components maxima in three sets of time-resolved spectra taken in natural and polarized light. A good fit of the experimental and calculated spectra was obtained when using the following maxima: 580 and 595 nm in the phycoerythrin region, 634 and 650 nm in the phycocyanin region, 660 and 680 nm in the allophycocyanin region. The area under curve of the Gaussian component vs. time gives the shape of rise and decay of emission of chromophores contributing to the given component. These kinetics were analysed using several model functions. The experimental excitation profile was convoluted with a multiexponential model individually or "globally" e.g. assuming the same lifetime values for the given species in all sets of spectra. The Foerster-Hauser types of two- and three-dimensional models we also convoluted with excitation profile and fitted to the decay of primarily excited species. The first acceptor decay can be described well by the Foerster-Hauser models or by a monoexponential function. The accuracy of fit in either case of three- and two-dimensional Foerster-Hauser function is similar. The fluorescence rise and decay of the next species in a donor-acceptor chain can be analysed in terms of two or three exponential functions. Obtained lifetimes of fluorescence are similar to those reported in literature. The results suggest that there are more than one chain of excitation donors and acceptors in the phycobilisomes of cyanobacteria Tolypothrix tenuis.     

A time-resolved fluorescence study of human copper-zinc superoxide dismutase

The intrinsic fluorescence decay of human Cu,Zn superoxide dismutase was measured by frequency-domain techniques. The protein consists of two subunits, each containing one tryptophan and no tyrosine residues. Using a synchrotron radiation source, which allows facile selection of the excitation wavelength, the dependence of the emission decay upon excitation was studied. No significant excitation wavelength effects were found. The two tryptophans contained in the dimer, although fully equivalent and exposed to solvent, showed a fluorescence decay that cannot be described by a single lifetime. Either two lifetimes, or one Lorentzian-shaped continuous distribution of lifetimes, are needed to obtain a good fit. Under identical experimental conditions, control experiments showed that N-acetyltryptophanamide, an analogue of tryptophanyl residues in proteins, decays with a single lifetime. The heterogeneous decay of tryptophan fluorescence in superoxide dismutase is interpreted as due to the presence of static and/or dynamic conformers in the protein that decay with different lifetimes. The two models of discrete lifetimes and continuous distribution of lifetimes are discussed with reference to measurements on holo- and apo-human superoxide dismutase.