RESUMEN
Influence of ionic (NaCl) and non-ionic (sorbitol) additives on structural transitions of cytochrome c was investigated by circular dichroism, optical and EPR spectroscopy. Transformations of cytochrome c, induced by the acidification of solution and temperature perturbation, were monitored in the heme pocket together with changes in the secondary structure. NaCl and sorbitol exhibited antagonistic effect on the acid-induced transition of the protein. Sorbitol enhanced the stability of native conformation while NaCl destabilized this state. The midpoints of acid-induced transitions in the axial coordination of heme as well as in the secondary structure occurred nearly at the same pH values. However, temperature-induced transitions in the unfolding of the secondary structure were almost coincidental with the cleavage of Met80-Fe bond only in the sorbitol solutions. In the salt solution the Met80-Fe bond was markedly more labile than the secondary structure.
Asunto(s)
Citocromos c/química , Cloruro de Sodio/química , Sorbitol/química , Animales , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Estabilidad de Enzimas , Caballos , Humanos , Concentración de Iones de Hidrógeno , Conformación Proteica , Espectrofotometría , TemperaturaRESUMEN
Temperature-gradient gel electrophoresis (TGGE) has been used to study the thermal unfolding of ferricytochrome c in low and high concentrations of acetic acid. It has been observed that the mobility of cytochrome c is a linear function of temperature when the system is characterized by a homogeneous population of conformation-state, single molecular species. Within the transition temperature range, the mobility clearly displays the characteristic sigmoidal shape describing the transitions of protein unfolding. The data obtained by TGGE were used to estimate the apparent thermodynamic parameters (enthalpy change deltaHvh and transition temperature Tm), associated with the transition of unfolding. The accuracy of the apparent thermodynamic parameters obtained by this method agrees within error limits with the values obtained by direct calorimetric measurements using differential scanning calorimetry (DSC).
Asunto(s)
Grupo Citocromo c/química , Electroforesis en Gel de Poliacrilamida/métodos , Calor , Conformación Proteica , Rastreo Diferencial de Calorimetría , Concentración de Iones de Hidrógeno , TermodinámicaRESUMEN
The surface tension behaviour of oxidised cytochrome c (cyt c) solution was characterised at various pH and ionic strength at the air/water interface. The pendant drop method employing digital image analysis of the drop shape was applied to the measurement of the surface tension. The adsorption properties of cyt c were utilised to study the protein conformation change effected by acidification and ionic strength. At high ionic strength, the saturated steady-state surface tension shows a cooperative change centred around 3.6 induced by a decrease in pH. Using spectroscopic experiments, the apparent pK of the acid-induced transition of horse cyt c from the native to the molten globular state is equal to 3.5. This fact indicates that the saturated steady-state surface tension is a parameter which might be used to monitor conformation changes of cyt c.
Asunto(s)
Grupo Citocromo c/química , Concentración de Iones de Hidrógeno , Concentración Osmolar , Oxidación-Reducción , Conformación Proteica , Soluciones , Tensión SuperficialRESUMEN
The adsorption properties of cytochrome c (cyt c) were characterized by surface tension measurements using the pendant-drop method employing the digital image analysis of the drop shape. The method was applied to the study of the protein conformation change due to acidification at low ionic strength. The observation of the saturated steady-state surface tension shows that decrease in pH induces its cooperative change centered around pH 2.5. This value is equal to the value of apparent pK of the acid-induced transition of the horse ferricyt c from a native state to the unfolded conformation. This indicates that the saturated steady-state surface tension is sensitive to the conformation of cyt c in bulk phase, and the pendant-drop method might be used to monitor changes in the tertiary structure of proteins.
Asunto(s)
Grupo Citocromo c/química , Conformación Proteica , Adsorción , Animales , Caballos , Concentración de Iones de Hidrógeno , Concentración Osmolar , Tensión Superficial , TemperaturaRESUMEN
The properties of the complex of ferricyt c with fluorosulfonated polyanion Nafion (as a representative 'hydrophobic' polyanion) have been studied by means of optical spectroscopy and differential scanning calorimetry. The addition of the polyanion to a solution of ferricyt c (pH 7.4) resulted in an expansion of the protein molecule characterized by a profound decrease in enthalpy of the thermal transition of ferricyt c. The conformational change of ferricyt c upon addition of Nafion was shown by a perturbation of the Met-80-heme iron bond and an apparent increase in the distance of Trp-59 from the heme. The conformational change in the heme region was also observed by examining the CD spectra. The conformational state of ferricyt c in a complex with Nafion was similar to that designated as state II by Hildebrandt (Hildebrandt, P. (1990) Biochim. Biophys. Acta 1040, 175-186) in the complex with negatively charged heteropolytungstates-a six-coordinated low-spin state with a destabilized structure of the heme crevice. The decrease in enthalpy of the thermal transition of ferricyt c, the spectral changes in absorbance and the CD spectra, together with an increase in Trp fluorescence induced by Nafion addition observed at high ionic strength, all point to the involvement of the non-coulombic interaction.
Asunto(s)
Grupo Citocromo c/química , Grupo Citocromo c/metabolismo , Polímeros de Fluorocarbono/química , Polímeros de Fluorocarbono/metabolismo , Animales , Rastreo Diferencial de Calorimetría , Polímeros de Fluorocarbono/farmacología , Caballos , Cinética , Miocardio , Conformación Proteica/efectos de los fármacos , Espectrofotometría , TriptófanoRESUMEN
The effect of saturated solutions of polyglutamate on the thermal stabilities of the Met-80-heme iron bond and of the ferricytochrome c as a whole were studied by absorption spectroscopy and differential scanning calorimetry at pH 7.0. According to spectral data the midtransition temperature of the cleavage of the sulfur-iron bond was 57.4 +/- 0.5 degrees C and 66.8 +/- 0.5 degrees C for cytochrome c and cytochrome c-polyglutamate complex, respectively. Addition of polyglutamate to cytochrome c at pH 7.0 alters the denaturation properties of the protein. As follows from DSC scans, the denaturation temperature for cytochrome c is decreased from 85.4 +/- 0.2 degrees C to 68.7 +/- 0.2 degrees C in the presence of the saturated amount of polyglutamate. The protein stability in terms of Gibbs energy change at protein unfolding amount to delta G(25 degrees C) = 22.7 +/- 2.7 and 32.0 +/- 2.2 kJ/mol, for cytochrome c and cytochrome c-polyglutamate complex, respectively, at pH 7.0. It is evident that polyglutamate increases the thermal stability of the sulfur-iron bond and decreases the denaturation temperature of the cytochrome c molecule as a whole. The complex thermal stability in terms of Gibbs energy is not lower than that of cytochrome c in the range of physiological temperatures.
Asunto(s)
Grupo Citocromo c/química , Grupo Citocromo c/efectos de los fármacos , Ácido Poliglutámico/farmacología , Animales , Rastreo Diferencial de Calorimetría , Estabilidad de Enzimas/efectos de los fármacos , Caballos , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína , Soluciones , Espectrofotometría , Temperatura , TermodinámicaRESUMEN
The effect of nucleotides on the structure and thermal stability of ferricytochrome c was studied by differential scanning calorimetry. The association of cytochrome c with ATP and ADP resulted in a decrease in the denaturation temperature of cytochrome c by 7 degrees C and 4 degrees C, respectively, at pH 7.0. AMP did not change the denaturation temperature of cytochrome c at pH 7.0. The ratio between van't Hoff and calorimetric enthalpy of denaturation accounts for the fact that cooperative denaturation of 3-4 molecules of cytochrome c occurred in the presence of ATP at the pH range from 5 to 9. ADP gave rise to the interaction of 2-3 molecules of ferricytochrome c at pH 6-7.5, and AMP did not affect the interaction of protein molecules. Cytochrome c alone also associated at pH 7.5-10. At physiological ionic strength, pH 7.0, only ATP induced an association of ferricytochrome c molecules. No intermolecular interaction of ferricytochrome c molecules was observed at concentrations of NaCl higher than 0.2 mol/l not even in the presence of ATP.
Asunto(s)
Grupo Citocromo c/metabolismo , Nucleótidos/farmacología , Nucleótidos de Adenina/farmacología , Animales , Rastreo Diferencial de Calorimetría , Citidina Trifosfato/farmacología , Grupo Citocromo c/química , Estabilidad de Medicamentos , Guanosina Trifosfato/farmacología , Caballos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Estructura Molecular , Desnaturalización Proteica/efectos de los fármacos , Temperatura , Termodinámica , Uridina Trifosfato/farmacologíaRESUMEN
The effects of heparin on the thermotropic properties of ferricytochrome c have been studied using high-sensitivity differential scanning calorimetry. Saturating concentrations of heparin at low ionic strength induced an important shift of the transition temperature Tm from 84.1 degrees C to 59.8 degrees C. This was accompanied by unusually large cooperativity of thermal denaturation of this complex, indicating strong intermolecular interactions between protein molecules. The destabilization of cytochrome c when mixed with heparin was not observed at high ionic strength, under which conditions complex was not formed.
Asunto(s)
Rastreo Diferencial de Calorimetría , Grupo Citocromo c/química , Grupo Citocromo c/metabolismo , Heparina/metabolismo , Calor , Concentración Osmolar , Desnaturalización Proteica , Cloruro de Sodio/farmacología , TermodinámicaRESUMEN
As detected by adiabatic differential scanning microcalorimetry, [2-(alkyloxy)-phenyl]-2-(1-piperidinyl)ethyl esters of carbamic acid (CnPPEECA, n is the number of carbon atoms in the alkyloxy substituent) with local anesthetic and antiarrhythmic activities interact with 1,2-dipalmitoyl-sn-glycero-3-[phosphorac-(1-glycerol)] model membranes (DPPG). CnPPEECAs form solid-like solutions with DPPG at low CnPPEECA concentrations and with short alkyloxy chain lengths (n < 4), while at higher concentrations and with longer alkyloxy chains (10 > or = n > or = 5) demixing and separation of CnPPEECA+DPPG clusters of unknown stoichiometry occurs in the gel phase. The temperature of the gel-liquid crystal phase transition Tm is depressed in the presence of CnPPEECA; the depression of Tm scaled for unity CnPPEECA concentration in the lipid phase indicates higher intrinsic perturbation activity of the charged form of CnPPEECA than that of the basic form of CnPPEECA. It is suggested that this might be caused by a deeper location of the basic form of CnPPEECA in the lipid bilayer.
Asunto(s)
Carbamatos , Membranas Artificiales , Modelos Biológicos , Fosfatidilgliceroles/química , Piperidinas , Anestésicos Locales , Antiarrítmicos , Rastreo Diferencial de Calorimetría , Espectroscopía de Resonancia por Spin del ElectrónRESUMEN
Absorption UV-VIS and pre-resonance Raman spectra of acidic cyt c solutions with a series of thiols added (thiophenol, n-propanethiol, isopropanethiol, L-cysteine, dithiothreitol, 2-mercaptoethanol, N-acetyl-L-cysteine, p-acetamidothiophenol, 2-mercaptoethanamine, thioglycolic acid and mercaptopropionic acid), are presented. Interactions of cyt c molecule with the thiols were studied with the aim to identify binding of the thiols with the cyt c heme as its iron axial ligands. Absorption and Raman spectra showed some correlation between maxima of 700 nm region absorption band (typical for Fe-S axial bond in cyt c heme) and also wave numbers of spin state marker and axial ligand sensitive Raman bands on one, and pKa constant values of appropriate thiols on the other hand. These results imply thiol replacement of Met-80 from axial bond with heme iron and suggest that the force of Fe-L-cysteine axial bond is very close to the native axial bond (Fe-Met) for cyt c in neutral solution.