Horse Heart Myoglobin Research Articles

Spectroscopic Study of the Interaction between Horse Heart Myoglobin and Zirconium(IV)-Substituted Polyoxometalates as Artificial Proteases.

A recent study [Angew. Chem. Int. Ed. 2015, 54, 7391-7394] has shown that horse heart myoglobin (HHM) is selectively hydrolyzed by a range of zirconium(IV)-substituted polyoxometalates (POMs) under mild conditions. In this study, the molecular interactions between the Zr-POM catalysts and HHM are investigated by using a range of complementary techniques, including circular dichroism (CD), UV/Vis spectroscopy, tryptophan fluorescence spectroscopy, and 1 H and 31 P NMR spectroscopy. A tryptophan fluorescence quenching study reveals that, among all examined Zr-POMs, the most reactive POM, 2:2 ZrIV -Keggin, exhibits the strongest interaction with HHM. 31 P NMR spectroscopy studies show that this POM dissociates in solution, resulting in the formation of a monomeric 1:1 ZrIV -Keggin structure, which is likely to be a catalytically active species. In the presence of ZrIV -POMs, HHM does not undergo complete denaturation, as evidenced by CD, UV/Vis, tryptophan fluorescence, and 1 H NMR spectroscopy. CD spectroscopy shows a gradual decrease in the α-helical content of HHM upon addition of ZrIV -POMs. The largest effect is observed in the presence of a large ZrIV -Wells-Dawson structure, whereas small ZrIV -Lindqvist POM has the least influence on the decrease in the α-helical content of HHM. In all cases, the Soret band at λ=409 nm is maintained in the presence of all examined Zr-POMs, which indicates that no conformational changes in the protein occur near the heme group.

The effect of the buffer solution on the adsorption and stability of horse heart myoglobin on commercial mesoporous titanium dioxide: a matter of the right choice.

Despite the numerous studies on the adsorption of different proteins onto mesoporous titanium dioxide and indications on the important role of buffer solutions in bioactivity, a systematic study on the impact of the buffer on the protein incorporation into porous substrates is still lacking. We here studied the interaction between a commercial mesoporous TiO2 and three of the most used buffers for protein incorporation, i.e. HEPES, Tris and phosphate buffer. In addition, this paper analyzes the adsorption of horse heart myoglobin (hhMb) onto commercial mesoporous TiO2 as a model system to test the influence of buffers on the protein incorporation behavior in mesoporous TiO2. N2 sorption analysis, FT-IR and TGA/DTG measurements were used to evaluate the interaction between the buffers and the TiO2 surface, and the effect of such an interaction on hhMb adsorption. Cyclic voltammetry (CV) and electron paramagnetic resonance (EPR) were used to detect changes in the microenvironment surrounding the heme. The three buffers show a completely different interaction with the TiO2 surface, which drastically affects the adsorption of myoglobin as well as its structure and electrochemical activity. Therefore, special attention is required while choosing the buffer medium to avoid misguided evaluation of protein adsorption on mesoporous TiO2.

Application of resonance Raman spectroscopy for interrogation of cryoradiolytically reduced oxygenated heme proteins

Resonance Raman (rR) spectroscopy is generally useful to interrogate unstable intermediates that arise in the enzymatic cycles of heme enzymes, such as peroxo-, hydroperoxo- and ferryl species. While it is typically difficult to trap these elusive intermediates in solution, the cryoradiolysis approach offers a way to successfully overcome these obstacles. The present work employs modified derivatives of horse heart myoglobin to demonstrate the methodology and utility of a combination of rR and cryoradiolysis for detection and structural characterization of Fe―O―O, Fe―O―O―H and FeO fragments of such reactive species. First, the effect of active site mutations on the Fe―O―O fragments was investigated; specifically, the His64Leu, Val68Ser and Ile107Ala in the distal pocket and Ser92Ala, Ser92Leu and His97Phe on the proximal side. The current work has permitted documentation of the wavenumbers of the ν(Fe―O) stretching modes of the relatively unstable dioxygen adducts of these mutants, some of them previously unreported. Significantly, applying the cryoradiolysis approach has yielded previously unavailable vibrational spectroscopic data for the peroxo-, hydroperoxo and ferryl derivatives of these mutants. Of the six mutations investigated, only two, the H64L and V68S, replacements had a significant effect on the structure and stability of these unstable intermediates. In addition to these manipulations of the heme active site environment, the study of myoglobins bearing modified hemes was also undertaken; namely, myoglobins reconstituted with mesoheme and diformyl deuteroheme. The results show that the heme equatorial electronic effects significantly influence the stability and internal modes of the Fe―O―O and FeO fragments of these unstable oxy intermediates. Copyright © 2016 John Wiley & Sons, Ltd.

Enhanced heme accessibility in horse heart mini-myoglobin: Insights from molecular modelling and reactivity studies

Mini-myoglobin (mini-HHMb) is a fragment of horse-heart myoglobin (HHMb) considered to be the prototype of the product encoded by the central exon of the HHMb gene. For this reason, mini-HHMb has been studied extensively showing that carbonylation and oxygenation properties of the ferrous form are similar to those of the full-length protein, while kinetics and thermodynamics of azide binding to the ferric form are significantly different from those of HHMb. To analyze the structure–function relationships in mini-HHMb and the role of conformational fluctuations in ligand accessibility, the molecular model of mini-HHMb has been built and refined by molecular dynamics simulations, and analyzed in parallel with that of full length HHMb. Moreover, imidazole binding parameters of ferric mini-HHMb and HHMb have been determined. Furthermore, structural data of ferric mini-HHMb and HHMb have been correlated with the imidazole and previously determined azide binding properties. Present results indicate that, despite the extensive trimming, the heme-α-helices E-F substructure is essentially unaltered in mini-HHMb with respect to HHMb. However, the heme-Fe atom displays an enhanced accessibility in mini-HHMb, which may affect both ligand association and dissociation kinetics.

EPR Analysis of Imidazole Binding to Methanosarcina acetivorans Protoglobin

Protoglobins form an unusual class of globin proteins with surprising structural features, such as a highly ruffled heme. Electron paramagnetic resonance spectra of the frozen solution of the ferric imidazole complex of Methanosarcina acetivorans protoglobin reveal the existence of two low-spin ferric heme complexes. Hyperfine sublevel correlation spectroscopy was used to study the imidazole complex of this protoglobin as well as the imidazole complex of horse heart myoglobin for comparison. The spin Hamiltonian values obtained for the dominant contribution of the imidazole-ligated protoglobin agree with a heme conformation in which the plane of the exogenous imidazole is turned more than 30° versus the imidazole plane of the proximal histidine ligand. In turn, the electron paramagnetic resonance data of the minority complex indicate a second conformer, in which the two ligand planes are aligned almost parallel to each other with a lengthened iron–nitrogen bond for the exogenous imidazole ligand.

Vibrational dynamics of thiocyanate and selenocyanate bound to horse heart myoglobin.

The structure and vibrational dynamics of SCN- and SeCN-bound myoglobin have been investigated using polarization-controlled IR pump-probe measurements and quantum chemistry calculations. The complexes are found to be in low and high spin states, with the dominant contribution from the latter. In addition, the Mb:SCN high spin complex exhibits a doublet feature in the thiocyanate stretch IR absorption spectra, indicating two distinct molecular conformations around the heme pocket. The binding mode of the high spin complexes was assigned to occur through the nitrogen atom, contrary to the binding through the sulfur atom that was observed in myoglobin derived from Aplysia Limacina. The vibrational energy relaxation process has been found to occur substantially faster than those of free SCN(-) and SeCN(-) ions and neutral SCN- and SeCN-derivatized molecules reported previously. This supports the N-bound configurations of MbNCS and MbNCSe, because S- and Se-bound configurations are expected to have significantly long lifetimes due to the insulation effect by heavy bridge atom like S and Se in such IR probes. Nonetheless, even though their lifetimes are much shorter than those of corresponding free ions in water, the vibrational lifetimes determined for MbNCS and MbNCSe are still fairly long compared to those of azide and cyanide myoglobin systems studied before. Thus, thiocyanate and selenocyanate can be good local probes of local electrostatic environment in the heme pocket. The globin dependence on binding mode and vibrational dynamics is also discussed.

EPR analysis of cyanide complexes of wild-type human neuroglobin and mutants in comparison to horse heart myoglobin

Electron paramagnetic resonance (EPR) data reveal large differences between the ferric (13C-)cyanide complexes of wild-type human neuroglobin (NGB) and its H64Q and F28L point mutants and the cyanide complexes of mammalian myo- and haemoglobin. The point mutations, which involve residues comprising the distal haem pocket in NGB, induce smaller, but still significant changes, related to changes in the stabilization of the cyanide ligand. Furthermore, for the first time, the full 13C hyperfine tensor of the cyanide carbon of cyanide-ligated horse heart myoglobin (hhMb) was determined using Davies ENDOR (electron nuclear double resonance). Disagreement of these experimental data with earlier predictions based on 13C NMR data and a theoretical model reveal significant flaws in the model assumptions. The same ENDOR procedure allowed also partial determination of the corresponding 13C hyperfine tensor of cyanide-ligated NGB and H64QNGB. These 13C parameters differ significantly from those of cyanide-ligated hhMb and challenge our current theoretical understanding of how the haem environment influences the magnetic parameters obtained by EPR and NMR in cyanide-ligated haem proteins.

Sub-100-ps structural dynamics of horse heart myoglobin probed by time-resolved X-ray solution scattering

Here we report sub-100-ps structural dynamics of horse heart myoglobin revealed by time-resolved X-ray solution scattering. By applying the time-slicing scheme to the measurement and subsequent deconvolution, we investigate the protein structural dynamics that occur faster than the X-ray temporal pulse width of synchrotrons (∼100ps). The singular value decomposition analysis of the experimental data suggests that two structurally distinguishable intermediates are formed within 100ps. In particular, the global structural change occurring on the time scale of 70ps is identified.

Probing the Unfolding of Myoglobin and Domain C of PARP-1 with Covalent Labeling and Top-Down Ultraviolet Photodissociation Mass Spectrometry

Ultraviolet photodissocation (UVPD) mass spectrometry was used for high mass accuracy top-down characterization of two proteins labeled by the chemical probe, S-ethylacetimidate (SETA), in order to evaluate conformational changes as a function of denaturation. The SETA labeling/UVPD-MS methodology was used to monitor the mild denaturation of horse heart myoglobin by acetonitrile, and the results showed good agreement with known acetonitrile and acid unfolding pathways of myoglobin. UVPD outperformed electron transfer dissociation (ETD) in terms of sequence coverage, allowing the SETA reactivity of greater number of lysine amines to be monitored and thus providing a more detailed map of myoglobin. This strategy was applied to the third zinc-finger binding domain, domain C, of PARP-1 (PARP-C), to evaluate the discrepancies between the NMR and crystal structures which reported monomer and dimer forms of the protein, respectively. The trends reflected from the reactivity of each lysine as a function of acetonitrile denaturation in the present study support that PARP-C exists as a monomer in solution with a close-packed C-terminal α helix. Additionally, those lysines for which the SETA reactivity increased under denaturing conditions were found to engage in tertiary polar contacts such as salt bridging and hydrogen bonding, providing evidence that the SETA/UVPD-MS approach offers a versatile means to probe the interactions responsible for conformational changes in proteins.

Dye-Affinity Partitioning of Acidic, Basic, and Neutral Proteins in Ionic Liquid-Based Aqueous Biphasic Systems

The partitioning behavior of model acidic (bovine serum albumin, BSA), basic (hen egg white lysozyme, LYS), and neutral (horse heart myoglobin, MYO) proteins in ionic liquid-based aqueous biphasic systems containing Reactive Red-120 dye has been reported. This study found that partition coefficients of the proteins depended on their chemical structures, pH of the aqueous phase, temperature, and composition of the aqueous biphasic system. It was found that partition coefficients of proteins in aqueous biphasic system containing RR-120 were in the order of LYS > MYO > BSA. Recovery of the hydrophilic ionic liquid, [C4mim]Br, was achieved by using hydrophobic ionic liquid, [C4mim]PF6.

Reduction of Ferrylmyoglobin by Hydrogen Sulfide. Kinetics in Relation to Meat Greening

The hypervalent meat pigment ferrylmyoglobin, MbFe(IV)═O, characteristic for oxidatively stressed meat and known to initiate protein cross-linking, was found to be reduced by hydrogen sulfide to yield sulfmyoglobin. Horse heart myoglobin, void of cysteine, was used to avoid possible interference from protein thiols. For aqueous solution, the reactions were found to be second-order, and an apparent acid catalysis could be quantitatively accounted for in terms of a fast reaction between protonated ferrylmyoglobin, MbFe(IV)═O,H(+), and hydrogen sulfide, H2S (k2 = (2.5 ± 0.1) × 10(6) L mol(-1) s(-1) for 25.0 °C, ionic strengh 0.067, dominating for pH < 4), and a slow reaction between MbFe(IV)═O and HS(-) (k2 = (1.0 ± 0.7) × 10(4) L mol(-1) s(-1) for 25.0 °C, ionic strengh 0.067, dominating for pH > 7). For meat pH, a reaction via the transition state {MbFe(IV)═O···H···HS}([symbol: see text]) contributed significantly, and this reaction appeared almost independent of temperature with an apparent energy of activation of 2.1 ± 0.7 kJ mol(-1) at pH 7.4, as a result of compensation among activation energies and temperature influence on pKa values explaining low temperature greening of meat.

Engineered metalloregulation of azide binding affinity and reduction potential of horse heart myoglobin

Metal ion binding to a previously reported variant of horse heart myoglobin (Lys45Glu/Lys63Glu) with a metal ion binding site on the surface of the protein that is adjacent to the haem binding site has been shown to influence ligand binding and electrochemical properties of the protein. For example, the K(d) (μM) for binding of azide to this variant decreases from 277 ± 9 to 32 ± 3 following addition of a saturating concentration of Mn(2+) (the value for the wild-type protein under the same conditions is 26 ± 1). Similarly, the midpoint reduction potential E(m) (mV vs. standard hydrogen electrode) increases from 9 to 40 in the presence of a saturating concentration of Mn(2+) (the value for the wild-type protein under the same conditions is 45 ± 2). These results demonstrate the potential value of engineered metal ion binding sites as a means of regulating the functional properties of even simple haem proteins.

A singular value decomposition approach for kinetic analysis of reactions of HNO with myoglobin

The reactions of several horse heart myoglobin species with nitrosyl hydride, HNO, derived from Angeli's salt (AS) and Piloty's acid (PA) have been followed by UV–visible, 1H NMR and EPR spectroscopies. Spectral analysis of myoglobin-derived speciation during the reactions was obtained by using singular value decomposition methods combined with a global analysis to obtain the rate constants of complex sequential reactions. The analysis also provided spectra for the derived absorbers, which allowed self-consistent calibration to the spectra of known myoglobin species. Using this method, the determined rate for trapping of HNO by metmyoglobin, which produces NO-myoglobin, is found to be 2.7×105M−1s−1 at pH7.0 and 1.1×105M−1s−1 at pH9.4. The reaction of deoxymyoglobin with HNO generates the adduct HNO-myoglobin directly, but is followed by a secondary reaction of that product with HNO yielding NO-myoglobin; the determined bimolecular rate constants for these reactions are 3.7×105M−1s−1 and 1.67×104M−1s−1 respectively, and are independent of pH. The derived spectrum for HNO-myoglobin is characterized by a Soret absorbance maximum at 423nm with an extinction coefficient of 1.66×105M−1cm−1. The rate constant for unimolecular loss of HNO from HNO-myoglobin was determined by competitive trapping with CO at 8.9×10−5s−1, which gives the thermodynamic binding affinity of HNO to deoxymyoglobin as 4.2×109M−1. These results suggest that the formation of HNO-ferrous heme protein adducts represents an important consideration in the biological action of HNO-releasing drugs.

P33 Determination of ferric sulfmyoglobin cyanide specie by resonance raman spectroscopy

Upon the exposure of the human body to high H2S concentrations, in the presence of oxidizing agents like O2and H2O2, a sulfur atom is incorporated into the heme group of the hemoglobin (Hb) generating a sulfheme derivatives. This sulfHb derivative promotes a condition known as Sulfhemoglobinemia, which decreases the Hb ability to execute its function of oxygen transportation. Thus, to further comprehend the formation of Sulfhemoglobinemia, we need to understand the mechanism that leads to the modification of the heme group and its implications in the heme chemical structure and stability. Regarding this, horse heart myoglobin (Mb) was used as model to determine a suitable method to produce the ferric sulfmyoglobin cyanide complex (CN-sulfheme) as a way to define the structural change of the heme group induced by the formation of this derivative. The formation of CN-sulfMb species was attempted by two different methods. In the first method, H2O2(l) was added to a metMb sample and then 1 μL of H2S(l) solution was added to produce the ferric-sulfMb Complex. This was followed by the addition of 3 M KCN solution to generate the CN-sulfMb (FeIII) derivative. In the second method, O2(g) was used instead of H2O2(l). Each reaction was followed by UV–vis Spectroscopy in the 300–700 nm region. The addition of KCNto the sample demonstrates a shift of the Soret band from 407 nm to 422 nm attributed to the CN-sulfMb (FeIII) complex. The metCN-SulfMb structure was evaluated by Resonance Raman Spectroscopy. The data demonstrated satellites bands around v4 (1354 cm−1 and 1390 cm−1) attributed to the distortion of the heme group due to the incorporation of the sulfur ring [1] . Both methods generated the same results, but the metCN-SulfMb species generated by the presence of O2 is more stable than the analog species general by the presence of H2O2.

Marked Difference in the Electronic Structure of Cyanide-Ligated Ferric Protoglobins and Myoglobin Due to Heme Ruffling

Electron paramagnetic resonance experiments reveal a significant difference between the principal g values (and hence ligand-field parameters) of the ferric cyanide-ligated form of different variants of the protoglobin of Methanosarcina acetivorans (MaPgb) and of horse heart myoglobin (hhMb). The largest principal g value of the ferric cyanide-ligated MaPgb variants is found to be significantly lower than for any of the other globins reported so far. This is at least partially caused by the strong heme distortions as proven by the determination of the hyperfine interaction of the heme nitrogens and mesoprotons. Furthermore, the experiments confirm recent theoretical predictions [Forti, F.; Boechi, L., Bikiel, D., Martí, M.A.; Nardini, M.; Bolognesi, M.; Viappiani, C.; Estrin, D.; Luque, F. J. J. Phys. Chem. B 2011, 115, 13771-13780] that Phe(G8)145 plays a crucial role in the ligand modulation in MaPgb. Finally, the influence of the N-terminal 20 amino-acid chain on the heme pocket in these protoglobins is also proven.

Human Neuroglobin Functions as a Redox-regulated Nitrite Reductase

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ∼2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

Quantification of Alefacept, an immunosuppressive fusion protein in human plasma using a protein analogue internal standard, trypsin cleaved signature peptides and liquid chromatography tandem mass spectrometry

Quantitative analysis of a therapeutic protein through use of surrogate proteotypic peptides was evaluated for the measurement of Amevive (Alefacept) in human plasma using liquid chromatography tandem mass spectrometry. Signature peptides were obtained through in silico and iterative tuning processes to represent Alefacept for quantification. Horse heart myoglobin was chosen as a protein analogue internal standard to compensate for errors associated with matrix effects and to track recovery throughout the entire sample pretreatment process. Samples were prepared for analysis by selective precipitation of the target proteins with pH controlled at 5.1 and heat denaturation at 45 °C followed by enzymatic digestion, dilution, and filtration. On-line extraction of the signature peptides was carried out using a Phenomenex Gemini C18 security guard column (4.0 mm × 2.0 mm) as a loading column and a Gemini C18 (100 mm × 2.1 I.D., particle size 5 μm) as the analytical (eluting) column. Tandem mass spectrometric detection was performed on a hybrid triple quadrupole linear ion trap equipped with electrospray ionization to positively ionize signature peptides for Alefacept and myoglobin. The method was linear for Alefacept (protein) concentrations between 250 and 10,000 ng/mL. Precision and accuracy for inter- and intra-assay for the lower limit of quantification was less than 20% (16.2 and 10.3, respectively). The method was validated according to current FDA guidelines for bioanalytical method validation.

Effects of Guanidination with Trypsin, Lys-C, or Glu-C Digestion on Mass Spectrometric Signal Intensity and Protein Sequence Coverage

The conventional peptide modification process of guanidination, in which the amino groups of lysine residues are converted to guanidino groups using O-methylisourea to create more basic homoarginine residues, is often used to improve the signal intensity of lysine-containing peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Here, we used three different protease enzymes (trypsin, Lys-C, and Glu-C) to evaluate the effects of guanidination on the MS signals of two enzymatically digested proteins. Horse heart myoglobin and bovine serum albumin were guanidinated either before or after digestion with trypsin, Lys-C, or Glu-C. The resulting peptides were subjected to MALDI-MS, and signal intensities and sequence coverage were systematically evaluated for each digest. Guanidination prior to Glu-C digestion improved sequence coverage for both proteins. For myoglobin, guanidination before enzymatic digestion with trypsin or Lys-C also enhanced sequence coverage, but guanidination after enzymatic digestion enhanced sequence coverage only with Lys-C. For albumin, guanidination either before or after Glu-C digestion increased sequence coverage, whereas pre- or post-digestion guanidination decreased sequence coverage with trypsin and Lys-C. The amino acid composition of a protein appears to be the major factor determining whether guanidination will enhance its MALDI-MS sequence coverage.

Pressure‐assisted tryptic digestion using a syringe

A simple and effective digestion method was developed using a syringe. A 3 mL syringe was used to apply a pressure of 6 atm to expedite tryptic digestion. Application of a pressure of 6 atm during digestion resulted in better digestion efficiency than digestion under atmospheric pressure. The protein peaks in the matrix-assisted laser desorption/ionization mass spectra of three model proteins (cytochrome c, horse heart myoglobin, and bovine serum albumin (BSA)) completely disappeared within 30 min at 37 degrees C under a pressure of 6 atm, with greater numbers of peptides observed in 30 min pressure-assisted digestion than in overnight atmospheric pressure digestion. This is mostly due to the miscleaved peptides. Similar sequence coverages were obtained for 30 min pressure-assisted digestion and overnight atmospheric pressure digestion of the three model proteins (92% vs. 88% for cytochrome c, 100% vs. 97% for horse heart myoglobin, and 53% vs. 53% for BSA).

Critical Temperature of Secondary Structural Change of Myoglobin in Thermal Denaturation up to 130 °C and Effect of Sodium Dodecyl Sulfate on the Change

The secondary structural change of horse heart myoglobin was examined in the thermal denaturation up to 130 degrees C. The original helicity of 82% gradually decreased to 67% with rise of temperature until 75 degrees C. Thereafter, it suddenly decreased to 24% at 90 degrees C and then slightly decreased to 14% at 130 degrees C. The helices of this protein were mostly destroyed between 75 and 100 degrees C. On the other hand, upon cooling to 25 degrees C from temperatures below 75 degrees C, the helicity completely recovered to the original value, but it did not after heating to temperatures above 80 degrees C. Thus, myoglobin maintains the reversibility of the structural change up to a temperature as high as 75 degrees C. This protein had another critical temperature around 90-100 degrees C in addition to 75 degrees C in the present thermal denaturation. Upon cooling to 25 degrees C after heating to temperatures above 80 degrees C, the extent of recovered helicity decreased with rise of temperature before cooling. The additive effect of sodium dodecyl sulfate (SDS) on the structural change of myoglobin differed below and above the critical temperature at 75 degrees C. In the temperature range below 75 degrees C where the structural change was reversible, the presence of SDS cooperated with the thermal denaturation to disrupt the structure. On the contrary, the presence of the surfactant more or less restrained the decrement of helicity at high temperatures above 85 degrees C. The helicity decreased and increased with an increase of SDS concentration upon cooling to 25 degrees C after heating to temperatures below 75 degrees C and after heating to temperatures above 85 degrees C, respectively. Then, upon cooling to 25 degrees C from any temperature, the helicity settled to a magnitude around 60% in the presence of the surfactant above 0.6 mM.