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PubMed2001

Methanol conversion in high-rate anaerobic reactors.

An overview on methanol conversion in high-rate anaerobic reactors is presented, with the focus on technological as well as microbiological aspects. The simple C1-compound methanol can be degraded anaerobically in a complex way, in which methanogens, sulfate reducing bacteria and homoacetogens interact cooperatively or competitively at substrate level. This interaction has large technological implications as it determines the final product of methanol mineralization, methane or carbon dioxide. The degradation route of methanol may be entirely different when environmental conditions change. Direct methanogenesis from methanol seems the predominant mineralization route under mesophilic conditions both in the absence and the presence of sulfate. Under thermophilic conditions methanol oxidation to carbon dioxide and hydrogen appears to play an important role. The UASB technology for mesophilic digestion of methanolic waste has presently reached full-scale maturity. The potential of methanol as feedstock for anaerobic processes is discussed.

Bacteria, Anaerobic↗
PubMed2002

[Effects of different methanol feeding strategy on hirudin production in high-density fermentation by recombinant Pichia pastoris].

Four different methanol feeding modes were evaluated in the hirudin production in high-density fermentation by Pichia pastoris. It was difficult to avoid methanol excessive in the broth with the feeding strategy only based on DO level. On the other hand, the fluctuation in methanol concentration was observed with methanol feeding strategy by off-line gas chromatography. However, the stable methanol concentration was perfectly achieved by the on-line monitoring with methanol sensor. The supply of energy was improved by feeding glycerol at a limited rate as well as methanol in the induction phase. Therefore, the high cell dry weight (162 g/L) and high hirudin activity (2.4 x 10(4) ATU/mL or 1.7 g/L) was obtained in the fed-batch fermentation of recombinant Pichia pastoris by methanol-glycerol mixed feeding.

Fermentation↗
PubMed2005

[Comparative study on the genotoxicity of gasoline-fueled vehicle exhaust and methanol-fueled vehicle exhaust].

OBJECTIVE: Comparing the genotoxicity of gasoline-fueled vehicle exhaust (gasoline exhaust for short) with that of methanol-fueled vehicle exhaust (methanol exhaust for short) so as to provide a scientific basis for replacement of gasoline by methanol as fuel in vehicle. METHODS: The MTT method was used first to evaluate the cytotoxicity of the two kinds of vehicle exhausts, and the concentration that had no obvious cytotoxicity would be used as the highest dose in the experiments. The A549 cells micronucleus test and single cell gel electrophoresis (SCGE) assay (comet assay) in vitro were applied to compare the genotoxicity of gasoline exhaust and methanol exhaust. RESULTS: The MTT results showed that the gasoline-fueled vehicle exhaust exerted stronger cytotoxicity to A549 cells in both 2 h and 24 h exposure times, compared with the methanol exhaust. In A549 cells micronucleus test in vitro, at doses 0.025, 0.05, 0.1 and 0.2 L/ml, the micronucleated cell (MNC) rates were 2.65%, 4.35%, 4.95% and 5.85% respectively, which were higher than those (1.30% and 1.35%) of controls (P<0.01). For the methanol-fueled vehicle exhaust, there was no significant difference in the rate of A549 cells micronucleus between the test groups and control group. In the comet assay, gasoline-fueled vehicle exhaust could induce A549 cells DNA damage. The rate of caudate cell and the length of DNA migration increased with the escalation of dosing level. However, the methanol-fueled vehicle exhaust did not show any DNA damage to A549 cells. CONCLUSION: The results demanstrate that gasoline-fueled vehicle exhaust can induce DNA and chromosome damage, it has a distinct genotoxicity, whereas the methanol-fueled vehicle exhaust does not show any potential genotoxicity in both tests.

DNA Damage↗
PubMed2005

[Comparative study on the mutagenicity of the gasoline-fueled vehicle exhaust and methanol-fueled vehicle exhaust].

OBJECTIVE: In order to compare the mutagenicity of the gasoline-fueled vehicle exhaust (gasoline exhaust for short) and the methanol-fueled vehicle exhaust (methanol exhaust for short), provide a scientific basis for replacement gasoline by methanol as fuel in vehicle. METHODS: The Ames assay and A549 cell micronucleus test in vitro were applied to compare the mutagenicity of gasoline-fueled vehicle exhaust and methanol-fueled vehicle exhaust. RESULTS: The results showed that the gasoline-fueled vehicle exhaust expressed evident mutagenicity in Ames assay (TA98 strain) with or without rat liver-derived metabolic activation system (S9). The mutagenicity increased obviously when S9 was used and showed a good dose-response relationship. However, the methanol-fueled vehicle exhaust did not show any mutagenic potential in Ames assay (TA98, TA100 strains) with or without rat liver-derived metabolic activation system (S9). In A549 cells micronucleus test in vitro, between 0.025 - 0.2JL/ml, the rate of A549 cells micronucleus could be induced by the gasoline-fueled vehicle exhaust. For the methanol-fueled vehicle exhaust, there was no significant differences in the rate of A549 cells micronucleus between the tested groups and control. CONCLUSION: The results strongly suggested that the gasoline-fueled vehicle exhaust had potential mutagenicity and the methanol-fueled vehicle exhaust did not show any potential mutagenicity. The results also provided a scientific basis for replacement gasoline by methanol as fuel in vehicle.

Animals↗
PubMed1990

[Spectral interactions of cytochrome P-450 with n-heptane and methanol].

Methanol interacts with cytochrome P-450 to produce the reversed type I spectral change. In the presence of type I substrate n-heptane, the methanol induced spectrum disappears without detectable effects of interaction suggesting that methanol is a very weak ligand for heme iron of cytochrome P-450. Methanol strongly lowers the apparent spectral dissociation constant (Ks app) of n-heptane binding with rat liver cytochrome P-450 both in control and C6-C9 petroleum fraction inhaled group. In control group, titration of cytochrome P-450 with methanolic solutions of n-heptane does not change the maximal spectral interaction (Amax) observed with pure n-heptane. However in the inhaled group during titration of induced cytochrome P-450 with methanolic solutions of n-heptane an additional type I spectral change is observed. Thus addition of "equivalent" amounts of methanol into the reference cuvette during titration with methanolic solutions overestimates the true magnitude of type I spectral change of cytochrome P-450 with n-heptane.

Animals↗
PubMed1990

The folding of staphylococcal nuclease in the presence of methanol or guanidine thiocyanate.

The effect of methanol on the folding of staphylococcal nuclease has been investigated. Equilibrium thermal unfolding transitions were monitored by fluorescence emission. The transition was very sensitive to the presence of methanol (at pH 7.0), the Tm decreased from above 50 degrees C for aqueous solution to below 0 degree C for 70% methanol. The transitions were fully reversible and conformed to two-state behavior. A linear relationship was observed between the hydrophobicity of the solvent and both the Tm and the change in delta G for unfolding. The effect of pH on the transition in 50% methanol at 0 degree C was essentially the same as for aqueous solution, with a cooperative transition in the vicinity of apparent pH (pH*) 4. The unfolding transition was determined as a function of guanidine thiocyanate in aqueous and 50% methanol solvents. The midpoints of the transitions were 0.30 and 0.20 M, respectively, at 2.1 degrees C. The kinetics of folding at 0 degree C were compared in aqueous, 50% methanol and 0.30 M guanidine thiocyanate solvents, by monitoring changes in the tryptophan fluorescence intensity. Triphasic kinetics for refolding in both aqueous and 50% methanol solutions were observed in stopped-flow experiments. In both solvent systems the slowest phase is ascribed to proline isomerization. The kinetics of refolding were monitored at subzero temperatures in 50% methanol at pH* 7.0 in manual mixing experiments. Biphasic kinetics were observed at temperatures between 0 and -35 degrees C. A third, faster phase, was inferred from the missing amplitude. The energies of activation were 20.0 and 17.2 kcal mol-1, respectively, for the two slower phases. At -33.8 degrees C, the observed pseudo first-order rate constants were 1.2 x 10(-3) and 2.1 x 10(-5) s-1. At temperatures above -35 degrees C, the sum of the observed amplitudes was essentially constant at 70-75% of the expected total amplitude. At lower temperatures the amplitude of the refolding reaction decreased, and the native state was not formed (unless the temperature was increased), due to the formation of a trapped intermediate state. This intermediate has circular dichroism and fluorescence properties consistent with a compact state with some molten globule characteristics.

Circular Dichroism↗
PubMed1995

[Ethanol-independent methanol elimination in chronic alcoholics].

72% of a collective of chronic alcoholics (DSM-III-R, ICD 9), who were admitted under the influence of alcohol in order to undergo alcohol withdrawal, showed a serum methanol concentration (SMC) above 10 mg/l. This level is usually considered to be the one for the detection of regular alcohol consumption. The SMC values were considerably higher in cases where alcoholic beverages with a higher methanol content were consumed rather than the ones lower in methanol. In the majority of patients a decrease of the methanol concentration could only be detected once an individually varying limit concentration of ethanol (0-0.62 g/kg) was reached. There were, however, a few exceptions where the elimination of methanol independent from the ethanol concentration could be seen. Contrasting the general collective, these 'ethanol independent' methanol eliminators showed a much higher serum level of ethanol and methanol at the time of admission. As a sign of addiction, all patients showed increased beta 60 values for ethanol and preferred high proof beverages, which at the same time have high methanol contents.

Adult↗
PubMed2005

Molecular modeling of phase behavior and microstructure of acetone-chloroform-methanol binary mixtures.

Force fields based on a Lennard-Jones (LJ) 12-6 plus point charge functional form are developed for acetone and chloroform specifically to reproduce the minimum pressure azeotropy found experimentally in this system. Point charges are determined from a CHELPG population analysis performed on an acetone-chloroform dimer. The required electrostatic surface for this dimer is determined from ab initio calculations performed with MP2 theory and the 6-31g++(3df,3pd) basis set. LJ parameters are then optimized such that the liquid-vapor coexistence curve, critical parameters, and vapor pressures are well reproduced by simulation. Histogram-reweighting Monte Carlo simulations in the grand canonical ensemble are used to determine the phase diagrams for the binary mixtures acetone-chloroform, acetone-methanol, and chloroform-methanol. The force fields developed in this work reproduce the minimum pressure azeotrope in the acetone-chloroform mixture found in experiment. The predicted azeotropic composition of x(CHCl3) = 0.77 is in fair agreement with the experimental value of x(CHCl3)expt = 0.64. The new force fields were also found to provide improved predictions of the pressure-composition behavior of acetone-methanol and chloroform-methanol when compared to other force fields commonly used for vapor-liquid equilibria calculations. NPT simulations were conducted at 300 K and 1 bar for equimolar mixtures of acetone-chloroform, acetone-methanol, and methanol-chloroform. Analysis of the microstructure reveals significant hydrogen bonding occurring between acetone and chloroform. Limited interspecies hydrogen bonding was found in the acetone-methanol or chloroform-methanol mixtures.

Journal Article↗
PubMed2006

Fourier transform infrared and quasielectron neutron scattering studies on the binding modes of methanol molecules in the confined spaces of HMCM-41 and HZSM-5: role of pore structure and surface acid sites.

Quasielastic neutron scattering (QENS) and Fourier transform infrared spectroscopic studies were carried out on methanol molecules adsorbed in HMCM-41 and HZSM-5 molecular sieves to monitor the effect of pore structure on their occluded state under the conditions of ambient temperature and 5-250 mbar pressures. The QENS results have shown that the pore geometry of the host matrix and the dipolar character of the adsorbate are together responsible for the binding state of guest molecules in the confining medium. Thus, neither translational nor free rotational motion was noticed for methanol molecules adsorbed in HZSM-5, in contrast to benzene and cyclohexane molecules of almost similar size that are reported to undergo a rotational motion under the identical conditions of loading (Phys. Chem. Chem. Phys. 2001, 3, 4449; 2003, 5, 3066). In the case of HMCM-41, a translational motion of occluded methanol molecules was clearly observed with a diffusion constant D approximately 1.5 x 10(-5) cm2 s(-1), as compared to a value of D approximately 2.6 x 10(-5) cm2 s(-1) for its liquid state. These results indicate that the adsorbed methanol experiences a considerable extent of supercooling due to capillary condensation in zeolitic pores, giving rise to formation of a metastable state even at room temperature. In HZSM-5, entrapped methanol exists in an almost solidlike state, whereas in HMCM-41, its density lies between that of the solid and the liquid phases. Infrared spectroscopic study conducted using deuterium-labeled adsorbate and host matrixes have given evidence for different kinds of interactions between the methanol molecules and the host matrix, depending upon the loading. For small loadings the internal hydroxy groups within the pore system get perturbed first, giving rise to formation of the methoxy groups. Multilayer adsorption and capillary condensation of methanol occur for a loading of 0.05 mmol per gram and above, within the pore system and also at the external surface, giving rise to a highly compressed state due to strong intermolecular bonding. At the same time, a considerable amount of exchange occurred between the hydroxy groups of the adsorbed methanol and those of the host matrix. Such exchange of hydroxy groups may play an important role in the catalytic properties of the porous aluminosilicates.

Journal Article↗
PubMed2006

Nanoparticle size effects on methanol electrochemical oxidation on carbon supported platinum catalysts.

The particle size effect observed on the performance of Pt/C electrocatalysts toward the methanol oxidation reaction (MOR) has been investigated with differential electrochemical mass spectrometry (DEMS). The investigation has been conducted under both potentiodynamic and potentiostatic conditions as research on methanol electrochemical oxidation is closely related to interest in direct methanol fuel cells. The particle size effect observed on the MOR is commonly regarded as a reflection of different Pt-CO and Pt-OH bond strengths for different particle sizes. This work focuses mainly on the mechanism of methanol dehydrogenation on platinum which is central to the problem of the optimization of the efficiency of methanol electro-oxidation by favoring the CO(2) formation pathway. It was found that the partitioning of the methanol precursor among the end products on supported platinum nanoparticles is strongly dependent on particle size distribution. Also, it is postulated that the coupling among particles of different sizes via soluble products must be considered in order to understand the particle size effects on the observed trends of product formation. An optimum particle size range for efficiently electro-oxidizing methanol to CO(2) was found between 3 and 10 nm, and loss in efficiency is mostly related to the partial oxidation of methanol to formaldehyde on either too small or too large particles. The possible reasons for these observations are also discussed.

Journal Article↗
PubMed2006

Electrooxidation of methanol on upd-Ru and upd-Sn modified Pt electrodes.

The electrochemical oxidation of methanol has been investigated on underpotentially deposited-ruthenium-modified platinum electrode (upd-Ru/Pt) and on underpotentially deposited-tin-modified platinum electrode (upd-Sn/Pt). The submonolayers of upd-Ru and upd-Sn on a Pt electrode increased the rate of methanol electrooxidation several times as large as that on a pure Pt electrode. The best performance for methanol electrooxidation was obtained on a ternary platinum based catalyst modified by upd-Ru and upd-Sn simultaneously. The influence of the submonolayers of upd-Ru adatoms and upd-Sn adatoms on the oxidation of methanol in acid has been investigated. The effect of Ru on methanol electrooxidation lies on the distribution of Ru adatoms on a Pt surface. It has been shown that as long as the amount of upd-Ru deposits were controlled in a proper range, upd-Ru deposits would enhance the methanol oxidation obtained on a Pt electrode at whichever deposition potential the upd-Ru deposits were obtained. The effects of tin are sensible to the potential range. The enhancement effect of upd-Sn adatoms for the oxidation of methanol will disappear as the electrode potential is beyond a certain value. It is speculated that there exists a synergetic effect on the Pt electrode as adatoms Ru and Sn participate simultaneously in the methanol oxidation.

Journal Article↗
PubMed1986

Utilization of Methanol plus Hydrogen by Methanosarcina barkeri for Methanogenesis and Growth.

Methanosarcina barkeri grew on methanol plus H(2). Both substrates were consumed in equimolar amounts. Growth was strictly dependent on the presence of acetate, which was required for the biosynthesis of cellular constituents. Only about 0.4% of the methane produced originated from acetate. By using deuterated methanol, it was demonstrated that methanogenesis from this compound under H(2) did not occur via oxidation of methanol to CO(2) and subsequent reduction but by direct reduction with H(2). Growth yields with methanol plus H(2) and with methanol alone were not significantly different: 2.8 g of cells per mol of methanol in mineral medium and 4.6 g of cells per mol of methanol in complex medium, respectively. Growth of M. barkeri on methanol plus H(2) depended strictly on the presence of sodium ions in the medium. In the presence of 50 mM K the K(s) for Na was 5 mM.

Journal Article↗
PubMed1998

Methanol promotes atmospheric methane oxidation by methanotrophic cultures and soils.

Two methanotrophic bacteria, Methylobacter albus BG8 and Methylosinus trichosporium OB3b, oxidized atmospheric methane during batch growth on methanol. Methane consumption was rapidly and substantially diminished (95% over 9 days) when washed cell suspensions were incubated without methanol in the presence of atmospheric methane (1.7 ppm). Methanotrophic activity was stimulated after methanol (10 mM) but not methane (1,000 ppm) addition. M. albus BG8 grown in continuous culture for 80 days with methanol retained the ability to oxidize atmospheric methane and oxidized methane in a chemostat air supply. Methane oxidation during growth on methanol was not affected by methane deprivation. Differences in the kinetics of methane uptake (apparent K(m) and V(max)) were observed between batch- and chemostat-grown cultures. The V(max) and apparent K(m) values (means +/- standard errors) for methanol-limited chemostat cultures were 133 +/- 46 nmol of methane 10 cells h and 916 +/- 235 ppm of methane (1.2 muM), respectively. These values were significantly lower than those determined with batch-grown cultures (V(max) of 648 +/- 195 nmol of methane 10 cells h and apparent K(m) of 5,025 +/- 1,234 ppm of methane [6.3 muM]). Methane consumption by soils was stimulated by the addition of methanol. These results suggest that methanol or other nonmethane substrates may promote atmospheric methane oxidation in situ.

Journal Article↗
PubMed1991

Methanol poisoning. A rodent model with structural and functional evidence for retinal involvement.

Methanol ingestion can lead to visual impairment, central nervous system dysfunction, or death. The extent of ocular involvement has been difficult to determine because the toxicity is restricted to humans and nonhuman primates due to species differences in methanol metabolism. A rodent model of methanol toxicity recently developed by us was used to evaluate retinal dysfunction in methanol poisoning. Formic acidemia and visual toxic reactions developed in methanol-intoxicated rats. Electroretinographic analysis indicated a significant early deficit in b-wave amplitude followed by a temporally delayed, lesser reduction in a-wave amplitude. Histologic evaluation of the eyes 60 hours after methanol administration revealed generalized retinal edema and vacuolation in the photoreceptors and retinal pigment epithelium. Ultrastructural examination showed swelling and disruption of the mitochondria in photoreceptor inner segments, optic nerve, and the retinal pigment epithelium. These studies document direct retinal involvement in this nonprimate model of methanol toxicity.

Animals↗
PubMed1994

Methanol-induced neural tube defects in mice: pathogenesis during neurulation.

A spectrum of cephalic neural tube defects was observed in near-term (gestation day [GD] 17) mouse fetuses following maternal inhalation of methanol at a high concentration (15,000 ppm) for 6 hr/day during neurulation (GD 7-9). Dysraphism, chiefly exencephaly, occurred in 15% of fetuses, usually in association with reduction or absence of multiple bones in the craniofacial skeleton and ocular anomalies (prematurely open eyelids, cataracts, retinal folds). Measurements of cerebrocortical width in grossly normal, methanol-exposed fetuses revealed significant semiquantitative differences in the thicknesses of the frontal cortex and its constituent layers (neuroepithelium, intermediate cortex/subventricular plate, and cortical layer 1) as well as apparent increases in subventricular plate cellularity relative to controls. Subsequently, the early morphogenesis of these neural changes was investigated in neurulating mouse embryos to define tissue-specific patterns of methanol-induced damage that lead to cephalic axial dysraphism. Following daily 6-hr maternal inhalations of 15,000 ppm methanol during GD 7-8, the cephalic neural fold margins were swollen, blunted, and poorly elevated on GD 8.5 and 9 relative to controls. Histopathology of exposed GD 8.5 embryos revealed microcephaly in association with reductions in the cell density and mitotic index of at least 47% in the cranial mesoderm. The mitotic index in the embryonic neuroepithelium was also reduced by 55%, and groups of neural crest cells were displaced to the neural folds dorsal to the foregut (relative to the more ventral location in the facial regions of control embryos). When examined on GD 9.5 and 10.5, maternal methanol exposure (15,000 ppm for 6 hr/day) during GD 7-9 resulted in stunting, delayed rotation, and microcephaly in over 90% of the affected embryos. Persistent patency of the anterior neuropore and prosencephalic hypoplasia were seen in > 40% and up to 90% of embryos, respectively. Shallow optic vesicles, stunted branchial arches, scoliosis, and hydropericardium were also observed. Many 10.5-day-old embryos were edematous. Occult dysraphism, recognized grossly by abnormally narrow cephalic conformation and histopathologically by the absence of mesoderm in the mesencephalon, was present in at least 21% of methanol-exposed embryos on GD 9.5 and 10.5. Nile blue vital dye staining of methanol-exposed embryos revealed no difference in dye accumulation between control and treated embryos on GD 8.5, 9.0, or 9.5. There were no apparent dysmorphogenic effects in control embryos at any stage of development.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Neurobehavioral effects of low-level methanol vapor exposure in healthy human volunteers.

Methanol-powered vehicles are being introduced in the United States as a solution to air pollution. This study assessed whether acute exposure to methanol vapor at the current industrial threshold limit value of 200 ppm for 4 hr has adverse effects on human neurobehavioral performance. Twenty-six healthy subjects (15 men, 11 women; ages 26-51 years) were exposed to methanol or water vapor for 4 hr while seated in a chamber. The subjects served as their own controls in a randomized, double-blind study design. The variables assessed were serum and urine methanol and formate levels; visual performance (color discrimination and contrast sensitivity); and neurophysiological (auditory evoked potentials) and neurobehavioral performances. Exposure to methanol increased serum concentrations and urinary excretions of methanol, but did not affect formate levels. Overall visual, neurophysiological, and neurobehavioral test outcomes were not significantly affected, unless certain between-subject variables are considered. Slight effects on P-300 amplitude and Symbol Digit testing were noted. We conclude that acute exposure of healthy people to low concentrations of methanol had little effect on these measures of neurobehavioral performance.

Administration, Inhalation↗
PubMed1995

Comparative toxicokinetics of methanol in the female mouse and rat.

The toxicokinetics of methanol in female CD-1 mice and Sprague-Dawley rats were examined to explore the possibility of species differences in the disposition of the compound. Mice received a single dose of 2.5 g/kg methanol either po (by gavage) or i.v. (as a 1-min infusion). Rats received a single oral dose of 2.5 g/kg methanol. As expected, the disposition of methanol was nonlinear in both species. Data obtained after i.v. administration of methanol to mice were well described by a one-compartment model with Michaelis-Menten elimination. Blood methanol concentration--time data after oral administration could be described by a one-compartment (mice) or two-compartment (rats) model with Michaelis-Menten elimination from the central compartment and biphasic absorption from the gastrointestinal tract. Kinetic parameters (Vmax for elimination, apparent volume of the central compartment [Vc], first-order rate constants for intercompartmental transfer [k12 and k21], and first-order absorption rate constants for fast [kAF] and slow [kAS] absorption processes) were compared between species. When normalized for body weight, mice evidenced a higher maximal elimination rate than rats (Vmax = 117 +/- 3 mg/hr/kg vs 60.7 +/- 1.4 mg/hr/kg for rats). The contribution of the fast absorption process to overall methanol absorption also was larger in the mouse than in the rat.

Administration, Oral↗
PubMed1996

The methanol-induced globular and expanded denatured states of cytochrome c: a study by CD fluorescence, NMR and small-angle X-ray scattering.

Methanol-induced conformational transitions of cytochrome c(cyt c) at acidic pH values were investigated with a combined use of far and near-UV CD, fluorescence, NMR spectroscopy and small-angle X-ray scattering. At pH 3.0 and 25 degrees C, two methanol-induced non-native states were characterized. First, addition of methanol up to 25% (v/v) induced a compact denatured conformer (I(M)). Further addition of methanol transformed this I(M) state into the expanded and highly helical denatured state (H). The existence of the I(M) state was shown by the discrepancy in transition curves obtained from the ellipticity at 222 nm, the ellipticity at 282 nm, the tryptophan fluorescence monitored at 350 nm and the native peak intensity of the (1)H NMR spectrum. These CD, fluorescence and NMR results showed that the I(M) state has no specific tertiary structure but has a secondary structural content and tryptophan environment similar to those in the native state. The radius of gyration of the I(M) state, 17.7 angstroms, obtained from the Guinier plot of the small-angle X-ray scattering data was significantly smaller than that of the acid-denatured state (30.1 angstroms) and was closer to that of the native state (14.6 angstroms), showing that the I(M) state is compact. The Kratky plot for the I(M) state exhibited a bell-shaped profile, indicating a globular conformation. These structural features indicate that the structure of the I(M) state is quite similar to that of the anion-induced molten globule state of this protein. Furthermore the alcohol-denatured state (H) of cyt C in 60% (v/v) methanol was structurally characterized. Though the H state had a helical content much higher than the native state monitored by far-UV CD spectroscopy, the radius of gyration, 31.7 angstroms, was similar to that of the acid-denatured state, showing that this H state is an expanded denatured state. The Kratky plot for the H state did not show a clear peak, indicating a chain-like conformation. Thus we conclude that the H state has an expanded and chain-like conformation with a high helical content. Finally, we constructed a phase diagram of cyt c involving the native, I(M), acid-denatured and H states against pH and the methanol concentration. The result indicates that the I(M) state is found in the pH range from 2.5 to at least 4.5 with a pH-dependent optimum methanol concentration of 10 to 40%.

Circular Dichroism↗