Intra- and intermolecular transfers of protein radicals in the reactions of sperm whale myoglobin with hydrogen peroxide

Reaction of sperm whale metmyoglobin (SwMb) with H2O2 produces a ferryl (MbFeIV=O) species and a protein radical and leads to the formation of oligomeric products. The ferryl species is maximally formed with one equivalent of H2O2, and the maximum yields of the dimer (28%) and trimer (17%) with 1 or 2 eq. Co-incubation of the SwMb Y151F mutant with native apoSwMb and H2O2 produced dimeric products, which requires radical transfer from the nondimerizing Y151F mutant to apoSwMb. Autoreduction of ferryl SwMb to the ferric state is biphasic with t = 3.4 and 25.9 min. An intramolecular autoreduction process is implicated at low protein concentrations, but oligomerization decreases the lifetime of the ferryl species at high protein concentrations. A fraction of the protein remained monomeric. This dimerization-resistant protein was in the ferryl state, but after autoreduction it underwent normal dimerization with H2O2. Proteolytic digestion established the presence of both dityrosine and isodityrosine cross-links in the oligomeric proteins, with the isodityrosine links primarily forged by Tyr151-Tyr151 coupling. The tyrosine content decreased by 47% in the dimer and 14% in the recovered monomer, but the yields of isodityrosine and dityrosine in the dimer were only 15.2 and 6.8% of the original tyrosine content. Approximately 23% of the lost tyrosines therefore have an alternative but unknown fate. The results clearly demonstrate the concurrence of intra- and intermolecular electron transfer processes involving Mb protein radicals. Intermolecular electron transfers that generate protein radicals on bystander proteins are likely to propagate the cellular damage initiated by the reaction of metalloproteins with H2O2.     

Protein partitioning at the isoelectric point: influence of polymer molecular weight and concentration and protein size

We report the partition coefficient, K(p') at the isoelectric point of lysozyme, chymotrypsinogen A, albumin, transferrin, and catalase in 64 different polyethylene(PEG)/ dextran(Dx)/water systems. We study the trends of the partition coefficient with protein type, polymer concentration, and polymer molecular weight. We find that the partition coefficient decreases with increasing tie line length for lysozyme, albumin, transferrin, and catalase for which K(p) is less than 1, but increases for chymotrysinogen for which K(p) is larger than 1. The effect of the tie line length on the partition coefficient is larger for the large proteins than for the small proteins. The partition coefficient decreases with increasing protein molecular weight except for lysozyme suggesting that lysozyme is present as a dimer or a trimer. The partition coefficient decreases with increasing PEG molecular weight, but the magnitude of the increase is larger for the smaller PEG molecular eights and tends to level of at high PEG molecular weight. The partition coefficient increases with increasing dextran (Dx) molecular weight for chymotrypsinogen but decreases for catalase. The partition coefficients of lysozyme, albumin, and transferrin increase with increasing Dx molecular weight from Dx 10(4) to Dx 1.1 x 10(5) and then slightly decrease from Dx 1.1 x 10(5) to Dx 5 x 10(5). The experimental results are analyzed using a statistical thermodynamics model. The experimental results are analyzed using a statistical thermodynamics model. The experiments suggest that protein partitioning at the isoelectric point in aqueous two-phase systems is strongly related to the size of the proteins and polymers. Finally, the impossibility of obtaining data completely independent of polymer concentration is emphasized.     

Mechanism of pppA(2'p5'A)n2'p5'AOH synthesis in extracts of interferon-treated HeLa cells

Treatment of HeLa cells with interferon results in the induction of an enzymatic activity designated 2'5'oligo(A) polymerase. The polymerase requires continuous presence of double-stranded RNA (dsRNA) for activity, since degradation of dsRNA abolishes synthesis of the oligomeric series pppA(2'p5'A)n. These oligonucleotides are formed initially at a constant rate with dimer synthesized faster than trimer, and the latter faster than tetramer. After 45 min, accumulation of the dimer declines whereas that of other oligomers still proceeds at a linear rate. These results suggest that an oligomer remains associated with the enzyme for possible consecutive additions of adenylate, since no significant accumulation of dimer precedes synthesis of trimer. The relative amounts of the different oligomers found at the end of a reaction may reflect an increasing probability of release as the oligomers are elongated. The accumulation of dimer, however, decreases when it becomes a substrate for adenylate addition; incorporation of isolated dimer into 2'5'-oligo(A) was directly shown. Other nucleotides with a blocked p5'A terminus, like A5'ppppp5'A and NADH, can serve as adenylate acceptors in the presence of dsRNA. The adenosine triphosphates 2'-dATP and 3'-dATP are not incorporated efficiently into 2'5'-oligo(A) and inhibit its synthesis.     

The mechanism of free base formation from DNA by bleomycin. A proposal based on site specific tritium release from Poly(dA.dU)

Poly(dA.dU), which is specifically tritiated at the 1'-, 2'- (ribo configuration), 3'-, or 4'-position of deoxyuridine, has been synthesized and the fate of the tritium has been determined upon degradation of the polymer by bleomycin, Fe(II), and O2. No tritium is labilized from the 1'-3H-labeled polymer as 3H2O; however, the resulting 3-(uridin-1'-yl)-2-propenal (uracil propenal) has the expected specific activity. The 2'-3H-labeled polymer affords 3H2O and no label in the uracil propenal. This result and the lack of solvent incorporation into the uracil propenal suggest that proton abstraction from C-2' to afford the trans-propenal is highly stereospecific. For the 3'-3H-labeled polymer, 3H2O is formed and the specific activity of the uracil propenal is identical to that of the deoxyuridine. This suggests that the labilization of the 3'-H is exclusively associated with free uracil formation. 3H2O is also formed from the 4'-3H-labeled polymer. These findings along with previous studies are consistent with the formation of uracil propenal and free uracil by the trapping of the initially formed 4'-radical species by O2 or by a monooxygen species, respectively.     

Catalytic properties of E. coli tryptophanase in a 50% aqueous solution of methanol and dimethylformamide

Tryptophanase from E. coli retains its ability to form quinonoid intermediate with L-alanine in water--methanol and water--dimethylformamide (1:1 v/v) solutions. Under these conditions the enzyme catalyzes decomposition of S-o-nitrophenyl-L-cysteine (SOPC) to o-nitrophenylthiol, pyruvate and ammonium ion. The enzyme's affinity for this substrate increases on going from water to water-organic solvents whereas the reaction rate decreases. In 50% methanol tryptophanase catalyzes the formation of L-tryptophan from indole and SOPC; in the mixture of 2H2O and C2H3O2H (1:1) the enzymatic isotope exchange of alpha-proton of L-phenylalanine with complete retention of configuration was observed.     

Singlet oxygen generation in the reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Singlet oxygen ((1)O(2)) generation in the reaction centers (RCs) of Rhodobacter sphaeroides wild type was characterized by luminescent emission in the near infrared region (time resolved transients and emission spectra) and quantified to have quantum yield of 0.03 +/- 0.005. (1)O(2) emission was measured as a function of temperature, ascorbate, urea and potassium ferricyanide concentrations and as a function of incubation time in H(2)O:D(2)O mixtures. (1)O(2 )was shown to be affected by the RC dynamics and to originate from the reaction of molecular oxygen with two sources of triplets: photoactive dimer formed by singlet-triplet mixing and bacteriopheophytin formed by direct photoexcitation and intersystem crossing.     

Molecular weight of the functional unit of human leukocyte, fibroblast, and immune interferons

There is good agreement between the target molecular weight and the known molecular weight of human leukocyte interferons (about 20,000). The target molecular weight of fibroblast interferon, 31,000 to 42,000, is significantly larger than the monomer molecular weight of 21,000 to 24,000, suggesting that the dimer may be the predominant active functional unit in solution. A range from 63,000 to 73,000 for the target molecular weight of several different fractions of immune interferon (including natural crude as well as the recombinant form) indicates that the functional form of the immune interferon may be a trimer or tetramer. Thus, these studies indicate that the functional unit of leukocyte interferon is the monomer, that of fibroblast interferon is a dimer, and that of immune interferon is probably a tetramer (or trimer).     

Re-examining the oligomerization state of macrophage migration inhibitory factor (MIF) in solution

The state of oligomerization of macrophage migration inhibitory factor (MIF, also known as glycosylation inhibiting factor, GIF) in solution has been variously reported as monomer, dimer, trimer, or mixtures of all three. Several crystal structures show MIF to be a trimer. Sedimentation velocity shows a recombinant human MIF sample is quite homogeneous, with 98% as a species with s(20,w)=3.07 S and D(20,w)=8.29 x 10(-7) cm(2)/s. Using the partial specific volume calculated from the amino acid composition these values imply a mass of 33.56 kDa, well above that of dimer, but also 9% below the trimer mass of 37.035 kDa. Sedimentation equilibrium data at loading concentrations from 0.01 to 1 mg/ml show unequivocally that the self-association is extremely tight. However, the apparent mass is 33.53 kDa [95% confidence 33.25-33.82], again 9% below that expected for 100% trimer. To examine the possibility this protein has an unusual partial specific volume, sedimentation equilibrium was also done in H(2)O/D(2)O mixtures, giving 0.765+/-0.017 ml/g rather than the calculated 0.735 ml/g. With this revised partial specific volume, the equilibrium and velocity data each give M=37.9+/-2.8 kDa, fully consistent with a strongly-associated trimeric quaternary structure.     

A reagentless optical biosensor based on the intrinsic absorption properties of peroxidase

During the reversible reaction between peroxidase (HRP) and H(2)O(2), several peroxidase intermediate species, showing different molecular absorption spectra, are formed which can be used for H(2)O(2) determination; when H(2)O(2) is generated in a previous enzymatic reaction, the substrate involved in this reaction can also be determined. On this basis, a new family of fully reversible reagentless optical biosensors containing HRP is presented; glucose determination is used as a model. The biosensor (which can be used for at least 6 months and/or more than 750 measurements) is prepared by HRP and glucose oxidase entrapment in a polyacrylamide gel matrix. A mathematical model (in which optical, kinetic and transport aspects are considered) relating the measured absorbance with the substrate concentration is also presented together with a simple methodology for characterization of this kind of biosensor. Regarding the optical model, the Kubelka-Mulk theory of reflectance does not give good results and the biosensors are better described by the Rayleigh theory of polymer solutions. Under working conditions, linear response ranges from 1.5x10(-6) to 3.0x10(-4)M glucose and CV was about 4%. This biosensor has been applied for glucose determination in fruit juices and synthetic serum samples without sample pretreatment.     

Catalase model systems. Decomposition of hydrogen peroxide catalysed by mesoferrihaem, deuteroferrihaem, coproferrihaem and haematoferrihaem.

The catalytic decomposition of H2O2 by deuteroferrihaem, mesoferrihaem, coproferrihaem and haematoferrihaem was studied as a model for the mechanism of action of catalase. For haematoferrihaem, anomalous but reproducible results were obtained, which could not be adequately explained. For each of the other ferrihaems studied, both monomeric and dimeric species catalysed decomposition, although the activity of monomer (aM) was much greater than that of dimer (aD). The pH variation of aD in the range 6.5--11 was consistent with an inverse dependence on [H+]1/2. The molecular mechanism whereby such a dependence could be achieved is not apparent. A study of the pH-dependence of aM in the range 6.5--11 revealed a linear inverse relationship with [H+]. This is interpreted in terms of attack by HO2- on ferrihaem monomer. The specific pH-independent rate constants for this reaction were in the order coproferrihaem greater than protoferrihaem greater than or equal to mesoferrihaem congruent to deuteroferrihaem. The order of magnitude of these rate constants is the same as that for catalysis by Fe(H2O)63+ and the second-order rate constant for decomposition of H2O2 by catalase. The implications on the mechanism of action of catalase are discussed.     

Detection by chemical cross-linking of interaction between high mobility group protein 1 and histone oligomers in free solution

Among the more abundant non-histone proteins is the high mobility group (HMG), with an unknown role in chromatin. We have investigated, by chemical cross-linking, the interaction of the protein HMG 1 with the histone dimer H2A X H2B and the histone tetramer (H3 X H4)2 in free solution. Cross-linking with dimethyl suberimidate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the cleavable cross-linker dimethyl-3,3'-dithiobispropionimidate, by two-dimensional electrophoresis reveals the existence of an interaction between HMG 1 and the histone dimer, and also between HMG 1 and the histone tetramer. In the case of the H2A X H2B dimer, the analysis of the patterns of the cross-linking products shows the presence of a trimer, (H2A X H2B) X HMG 1, and of another oligomer of higher molecular weight which also contains H2A X H2B and HMG 1. Non-histone HMG 1 has been found to interact with (H3 X H4)2, both by cross-linking kinetics and also by gel permeation chromatography, displaying a stoichiometry of one HMG 1/histone tetramer. The results have been interpreted as indicating the existence of an interaction between HMG 1 and both oligomers through two different binding sites.     

Metabolism of dietary procyanidins in rats

Procyanidins are major dietary polyphenols made of elementary flavan-3-ol (epi)catechin units. They have antioxidant properties and may contribute to health benefits in humans, but little is known about their metabolic fate. We compared here the metabolism of procyanidin dimer B3, trimer C2, and polymer isolated from willow tree catkins to that of catechin monomer in rats. These compounds were administered in the rat diet (0.1%, w/w) for 5 d and their metabolites estimated in 24 h urine. In rats fed procyanidins, neither parent compound nor catechin derivatives could be detected in contrast to animals fed catechin monomer, which excreted large amounts of catechin and its 3'-O-methylated form (25.7 +/- 0.6%). On the other hand, 16 metabolites of microbial origin were detected and identified as phenylvaleric, phenylpropionic, phenylacetic, and benzoic acid derivatives. Their total yields significantly decreased from the catechin monomer (10.6 +/- 1.1%) to the procyanidin dimer (6.5 +/- 0.2%), trimer (0.7 +/- 0.1%), and polymer (0.5 +/- 0.1%). Therefore, the degree of procyanidin polymerization has a major impact on their fate in the body characterized by a poor absorption through the gut barrier and a limited metabolism by the intestinal microflora as compared to catechin. This will have to be considered to explain the health effects of procyanidins. The contribution of their microbial metabolites should also be further investigated.     

An octamer of core histones in solution: central role of the H3-H4 tetramer in the self-assembly.

The association of histones H2A, H2B, H3, and H4 in solution has been studied. In 2 M NaCl and at neutral pH they can assemble in a complex in which each histone is present in equimolar amounts. The complex has a weight average molecular weight of 98,000 (+/- 3700) and a sedimentation coefficient (so20,w) of 4.8. The value of the weight average molecular weight and the histone stoichiometry indicate that the complex is an octamer. The pairs of histones H2A,H2B and H3,H4 studied separately under identical conditions only associated as equimolar complexes consistent with dimeric and tetrameric structures, respectively. The stability of the core histone octamer is a function of the ionic strength, pH, and concentration of protein. The octamer dissociates by losing dimers of H2A,H2B until the main complexes existing in solution are the H3.H4 tetramer and the H2A.H2B dimer. This process is reversible upon reestablishing the original conditions.     

A spectroscopic characterization of a monomeric analog of copper,zinc superoxide dismutase

A mutated protein of human Cu(II)₂Zn(II)₂ SOD in which residues Phe50 and Gly51 at the dimer interface were substituted by Glu's, thus producing a monomeric species, has been characterized by electronic absorption spectroscopy, EPR, relaxivity and¹H NMR techniques. Such substitutions and/or accompanying remodeling and exposure of the dimer interface to solvent, alter the geometry of the active site: increases in the axiality of the copper chromophore and the Cu-OH₂ distance have been observed. The affinity of both metal binding sites for Co(II) is also altered. The observed NMR parameters of the Co(II) substituted derivative have been interpreted as a function of the decrease of rotational correlation time as a consequence of the lower molecular weight of the mutated protein. Sharper NMR signals are also obtained for the reduced diamagnetic enzyme. Results are consistent with an active site structure similar to that observed for the dimeric analog Thr137Ile characterized elsewhere. An observed proportional decrease in enzymatic activity and affinity for the N₃-anion suggests the importance of electrostatic forces during substrate docking and catalysis.     

Altered expression of HES-1, BETA2/NeuroD, and PDX-1 is involved in impaired insulin synthesis induced by glucocorticoids in HIT-T15 cells

Rat tail tendon (RTT) collagen has been reacted with a homologous series of chromium(III) complexes viz., (H2O)(4)Cr(OH)(2)Cr(H2O)(4+)(4) 1 (dimer), Cr(3)(OH)(4)(H2O)(5+)(9) 2 (trimer), and Cr(4)(OH)(4)(O2)(H2O)(4+)(12) 3 (tetramer), and the structural alterations brought about by these complexes have been investigated through atomic force microscopy (AFM) and circular dichroism (CD) studies. Examination of Cr(III)-treated tendons using AFM revealed changes in the D-periodicity of collagen, which may arise due to differences in the topological distribution of various Cr(III) complexes. Evidence for organisation of monomeric collagen into quarter staggered fibrils in the presence of Cr(III) dimer, 1, has been obtained. The quaternary structural changes induced by chromium in the protein have been correlated to the conformational changes of collagen in the absence of denaturation.     

Formation of soluble fibrin oligomers in purified systems and in plasma.

The kinetic parameters for release of fibrinopeptide A (FPA) from human fibrinogen by thrombin are: Km = 2.3 X 10(-6)M and Vmax. = 1.1 X 10(-10)mol of FPA/s per unit of thrombin; for fibrin formation, Km is similar to that for FPA release, but, the conditions of the present study, Vmax. was approximately half of that for FPA release. The formation of fibrin polymer before the sol-gel transition was studied by gel-permeation chromatography combined with effluent analysis for fibrinogen antigen and residual FPA. Polymer formation in purified fibrinogen incubated with thrombin proceeded as a bimolecular association of exposed sites in a manner predicted by probability calculations and assuming random FPA cleavage. Each oligomer consisted of n molecules of fibrin monomer and two fibrinogen molecules, each of the latter lacking one FPA molecule, i.e. each oligomer, regardless of molecular size, retains two FPA molecules. The addition of 5 mM-CaCl2 to the reaction mixture changed the rate of polymer formation, so that dimer was no longer the prevalent oligomer; in the presence of Ca2+, the trimer was the oligomer in highest concentration. The polymers formed in the presence of calcium were similar in composition to those without, i.e. 2 mol of FPA/mol of oligomer. EDTA-treated plasma samples incubated for short periods of time, 30s or less, with thrombin ranging in concentration up to 1 N.I.H. unit/ml did not form clots during the 10-15 min period of observation until they were applied to the column, though a large proportion of the available FPA was cleaved (maximum 45%). The soluble polymers in plasma were mostly of the high-Mr variety (tetramer and greater); these high-Mr polymers contained less than 2 mol of FPA/mol of polymer, whereas dimer and trimer in plasma were similar to those in the purified systems, i.e. 2 mol of FPA/mol.     

Human liver alcohol dehydrogenase: purification, composition, and catalytic features.

Alcohol dehydrogenase has been purified from human liver by affinity chromatography. Ultracentrifugation, Sephadex G-200 chromatography, and amino acid analyses of multiple preparations demonstrate homogeneity of molecular weight. Sodium dodecyl sulfate disc gel electrophoresis reveals a single species of molecular weight 42 000. Based on a molecular weight of 85 000 for the dimer obtained from the amino acid composition and a molar absorptivity of A280nm0.1% = 0.58, the enzyme contains 3.6-4.2 g-atoms of zinc, as determined by emission spectrography, microwave-induced emission, and atomic absorption spectrometry. Inhibition by o-phenanthroline, (ethylenedinitrilo)tetraacetic acid, and alpha,alpha'-bipyridine demonstrates that zinc is essential to enzymatic function. Detailed kinetic analyses using primary alcohols of the homologous series CH3(CH2)nOH, n = 0-5, and the corresponding aldehydes as substrates show that KM values become smaller as n increases. This suggest that hydrophobic interactions play a role in substrate binding. The availability of well-defined preparations of human liver alcohol dehydrogenase now allows definitive genetic and functional studies of this enzyme to elucidate human ethanol metabolism.     

Substitution, cooperative, and solvent effects on π pnicogen bonds in the FH2P and FH2As complexes

Ab initio calculations have been carried out to study the substitution effect on the π pnicogen bond in ZH(2)P-C(2)HM (Z?=?H, H(3)C, NC, F; M?=?H, CH(3), Li) dimer, cooperative effect of the π pnicogen bond and hydrogen bond in XH-FH(2)Y-C(2)H(4) (X?=?HO, NC, F; Y?=?P and As) trimer, and solvent effect on the π pnicogen bond in FH(2)P-C(2)H(2), FH(2)P-C(2)H(4), FH(2)As-C(2)H(2), and FH(2)As-C(2)H(4) dimers. The interaction energy of π pnicogen bond increases in magnitude from -1.51?kcal?mol(-1) in H(3)P-C(2)H(2) dimer to -7.53?kcal?mol(-1) in FH(2)P-C(2)HLi dimer at the MP2/aug-cc-pVTZ level. The π pnicogen bond is enhanced by 12-30?% due to the presence of hydrogen bond in the trimer. The π pnicogen bond is also enhanced in solvents. The natural bond orbital analysis and symmetry adapted perturbation theory (SAPT) were used to unveil the source of substitution, cooperative, and solvent effects.     

Inactivation of Coprinus cinereus peroxidase by 4-chloroaniline during turnover: comparison with horseradish peroxidase and bovine lactoperoxidase

The peroxidase from Coprinus cinereus (CPX) catalyzed oxidative oligomerization of 4-chloroaniline (4-CA) forming several products: N-(4-chlorophenyl)-benzoquinone monoamine (dimer D), 4,4'-dichloroazobenzene (dimer E); 2-(4-chloroanilino)-N-(4-chlorophenyl)-benzoquinone (trimer F); 2-amino-5-chlorobenzoquinone-di-4-chloroanil (trimer G); 2-(4-chloroanilino)-5-hydroxybenzoquinone-di-4-chloroanil (tetramer H) and 2-amino-5-(-4-chlroanilino)-benzoquinone-di-4-chloroanil (tetramer 1). In the presence of 4-CA and H2O2, CPX was irreversibly inactivated within 10 min. Inactivation of CPX in the presence of H2O2 was a time-dependent, first-order process when the concentration of 4-CA was varied between 0 and 2.5 mM. The apparent dissociation constant (Ki) for CPX and 4-CA was 0.71 mM. The pseudo-first order rate constant for inactivation (k(inact)), was 1.15 x 10(-2) s(-1). Covalent incorporation of 20 mole 14C-4-CA per mole of inactivated CPX was observed. The partition ratio was about 2200 when either 4-CA or H2O2 was used as the limiting substrate. These results show that 4-CA is a metabolically activated inactivator (i.e. a suicide substrate). Unmodified heme and hydroxymethyl heme were isolated from native, 4-CA-inactivated and H2O2-incubated CPX. Inactivation resulted in significant losses in both heme contents. Analysis of tryptic peptides from 4-CA-inactivated CPX by MALDI-TOF/ MS and UV-VIS spectrophotometry suggested that trimer G and tetramer H were the major 4-CA derivatives that were covalently bound, including to a peptide (MGDAGF-SPDEVVDLLAAHSLASQEGLNSAIFR) containing the heme binding site. These studies show that heme destruction and covalent modification of the polypeptide chain are both important for the inactivation of CPX. These results were compared with similar studies on 4-CA-inactivated horseradish peroxidase (HRP) and bovine lactoperoxidase (LPO) during the oxidation of 4-CA.     

H2O2-mediated cross-linking between lactoperoxidase and myoglobin: elucidation of protein-protein radical transfer reactions

The H(2)O(2)-dependent reaction of lactoperoxidase (LPO) with sperm whale myoglobin (SwMb) or horse myoglobin (HoMb) produces LPO-Mb cross-linked species, in addition to LPO and SwMb homodimers. The HoMb products are a LPO(HoMb) dimer and LPO(HoMb)(2) trimer. Dityrosine cross-links are shown by their fluorescence to be present in the oligomeric products. Addition of H(2)O(2) to myoglobin (Mb), followed by catalase to quench excess H(2)O(2) before the addition of LPO, still yields LPO cross-linked products. LPO oligomerization therefore requires radical transfer from Mb to LPO. In contrast to native LPO, recombinant LPO undergoes little self-dimerization in the absence of Mb but occurs normally in its presence. Simultaneous addition of 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and LPO to activated Mb produces a spin-trapped radical electron paramagnetic resonance signal located primarily on LPO, confirming the radical transfer. Mutation of Tyr-103 or Tyr-151 in SwMb decreased cross-linking with LPO, but mutation of Tyr-146, Trp-7, or Trp-14 did not. However, because DBNBS-trapped LPO radicals were observed with all the mutants, DBNBS traps LPO radicals other than those involved in protein oligomerization. The results clearly establish that radical transfer occurs from Mb to LPO and suggest that intermolecularly transferred radicals may reside on residues other than those that are generated by intramolecular reactions.