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The self-association behavior of the Eph-kinases SAM domain has been studied in phosphate buffer, pH 7.4, containing 0.14 M NaCl using concentration-dependent sedimentation equilibrium experiments. Only weak interactions typical for a monomer-dimer equilibrium up to at least 12 mg/mL were observed. Such concentrated solutions require a consideration of the non-ideality expressed by virial coefficients. A special centrifuge equation was used for the global analysis to estimate equilibrium constants based on the thermodynamic activities of the reactants. When neglecting this, the parameters deviate by about 20%. Association constants for dimerization of the EphB2-SAM domain vary between 163 M(-1) at 10 degrees C and 395 M(-1) at 32 degrees C, indicating hydrophobic forces are involved in the dimerization process. In solutions of about 12 mg/mL, less than 50% dimers are in solution and higher oligomers can be excluded.

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Analysis of protein-protein interactions in highly concentrated solutions requires a consideration of the non-ideality in such solutions which is expressed by the virial coefficients. Different equations are presented to estimate effects of the thermodynamic non-ideality on the macromolecular interaction of self-associating proteins in sedimentation equilibrium experiments. Usually the influence of thermodynamic non-ideal behavior are described by concentration power series. The convergence of such power series is limited at high solute concentration. When expressing the thermodynamic non-ideality by an activity power series this disadvantage can be minimized. The developed centrifuge equations are the basis for a global analysis to estimate equilibrium constants and the corresponding thermodynamic activities of the reactants. Based on fit analysis of synthetic concentration profiles it was established that marked deviations from the expected association constants are observed for proteins with strong association forces between solute molecules. Considerable differences were also observed in weakly interacting systems. This was due to the excluded volume of the protein which is similar in magnitude to the binding constant. For interactions with moderate affinities values extremely close to the true binding values were obtained, as confirmed by experimental results with concanavalin A.

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This paper presents a modified method to determine experimentally the second virial coefficient of protein solutions by sedimentation equilibrium experiments. The improvement is based on the possibility of fitting simultaneously up to seven radial concentration distribution curves of solutions with different loading concentrations. The possibility of precise determination of the second virial coefficient allows estimation of the net charge and the excluded volume of a monomeric protein. Application of the method is demonstrated for lysozyme and ovalbumin. In 0.1 M sodium acetate buffer, pH 4.5, the second virial coefficient of hen egg white lysozyme amounts to 24 +/- 1 ml/g. Analysis based on spherical particle theory yield an excluded volume of 3.5 ml/g and a charge dependent value of 20.5 ml/g which is induced by a net charge number of 14.1 +/- 1. Under low salt conditions self-association processes on lysozyme are unfavorable due to electrostatic repulsion. To overcome these repulsive contributions, either a shift to neutral pH or addition of at least 2% NaCl is necessary. In this way the charge dependent contribution decreases below the value responsible for the excluded volume and allows crystallization of the protein. Similar effects can be observed with ovalbumin. The high virial coefficient observed at pH 8.5 is induced by the high net charge number of 27 +/- 1.

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Sedimentation and diffusion coefficients are important parameters to describe size and shape of macromolecules in solution. The data can be obtained from sedimentation velocity experiments by a nonlinear fitting procedure using approximate solutions for the Lamm equation. Here, we present a modification of such a model function that was originally proposed by Fujita [H. Fujita, Mathematical Theory of Sedimentation Analysis, Wiley, New York, 1962]. The extended model function is well suitable to study low molecular mass compounds. The improvement of this solution given here is based on using an adjustable value for the explicit integration variable, z, the reduced radius. This modification leads to more accurate sedimentation and diffusion coefficients compared to using a constant value of 0.5 as used by Fujita. The advantage of our modification was demonstrated by the analysis of noise-free curves calculated using the finite element method, as well as experimental curves obtained for the peptides angiotensin I and II. The relatively low sedimentation and diffusion coefficients found for both substances indicate that the peptides exist as extended chains of about 3.65 nm (angiotensin I) or 3.04 nm length (angiotensin II) in solution. The lack of higher-order structure of the peptides that was derived also from CD spectra might facilitate receptor binding, and could be one reason for the fast proteolytic digestion of the free peptides.

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Binding of heptameric GroES to the tetradecameric chaperonin GroEL in the absence or presence of nucleotides was investigated by analytical ultracentrifugation. In the absence of nucleotides, the association constant for the binding of GroES to GroEL, K1, was found to be approximately equal to 3 x 10(5) M(-1). The binding of a second GroES heptamer with only one-fourth the affinity of the first one can be neglected at subequimolecular concentrations relative to GroEL. Under these conditions, mainly an asymmetric "bullet"-shaped complex is formed [see also Schmidt et al. (1994) Science 265, 656-659]. In the presence of ADP or ATP analogues such as ATP-gamma-S or AMP-PNP, the affinity to bind GroES increases by at least 2 orders of magnitude depending on the nucleotide concentration. With increasing GroES:GroEL ratios in the presence of 1 mM ATP analogue, up to two GroES oligomers were bound to one GroEL oligomer, forming the symmetrical "American football"-shaped complex with apparently high affinity for the first GroES ring and considerably lower for the second one. These are the first results that provide an accurate and quantitative description of the equilibrium between asymmetrical and symmetrical complexes at relatively high concentrations of GroEL and GroES that are proposed to exist in vivo. We suggest that the increased affinity of GroEL for GroES plays a role in releasing substrate proteins from the central cavity of GroEL after folding under "non-permissive" conditions.

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Many of the functions of biological macromolecules are based on specific interactions. Extended concentration dependent studies of sedimentation coefficients or molecular masses of biopolymers are highly useful for describing the different kinds of association phenomena. These studies allow one to determine the partial concentrations of monomers and associates or reactants and complexes in self-associating systems or heterologous associations, respectively. Furthermore, in combination with corresponding measurements of biological activity these data allow one to estimate the individual activity parameters of components involved in equilibrium processes. The study of self-association and heterologous association using analytical ultracentrifugation, some recent developments therein, and its application to different examples are outlined here.

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A new method for the direct molecular mass determination from sedimentation velocity experiments is presented. It is based on a nonlinear least squares fitting procedure of the concentration profiles and simultaneous estimation of the sedimentation and diffusion coefficients using approximate solutions of the Lamm equation. A computer program, LAMM, was written by using five different model functions derived by Fujita (1962, 1975) to describe the sedimentation of macromolecules during centrifugation. To compare the usefulness of these equations for the analysis of hydrodynamic results, the approach was tested on data sets of Claverie simulations as well as experimental curves of some proteins. A modification for one of the model functions is suggested, leading to more reliable sedimentation and diffusion coefficients estimated by the fitting procedure. The method seems useful for the rapid molecular mass determination of proteins larger than 10 kDa. One of the equations of the Archibald type is also suitable for compounds of low molecular mass, probably less than 10 kDa, because this model function requires neither the plateau region nor a meniscus free of solute.

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Adrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4-114 and 4-108, were studied by high-sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 degrees C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change delta Cp = 7.28 +/- 0.67 kJ/mol/K, except H56R. The amino acid replacement H56R produces substantial local changes in the region around positions 56 and Y82, as indicated by reduced heat capacity change (delta Cp = 4.29 +/- 0.37 kJ/mol/K) and enhanced fluorescence. Deletion mutant 4-108 is apparently more stable than the wild type, as judged by higher specific denaturation enthalpy and resistance toward proteolytic degradation. No simple correlation between conformational stability and functional properties could be found.

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The CO-binding reaction of cytochrome P-450cam bound with (1R)-camphor and (1S)-camphor are compared in the temperature region of 210-260 K using time-resolved Fourier-transform infrared spectroscopy with the CO stretch vibration as spectroscopic probe. For (1S)-camphor as substrate the association of CO is slowed down by a factor of 2, while the dissociation is accelerated by a factor of 3. The CO complex for the (1S)-camphor-bound P-450 is less stabilized (deltaG=-22 kJ/mol) compared to the natural substrate (1R)-camphor (deltaG=-30 kJ/mol). The data are interpreted by a smaller change of the mobility of the (1S)-camphor due to CO binding as compared to (1R)-camphor, which would indicate a higher mobility of (1S)-camphor already in the CO free reduced form of P-450cam. The higher mobility of (1S)-camphor in the heme pocket might explain the increased uncoupling rate (hydrogen peroxide formation) of 11% [Maryniak et al. (1993) Tetrahedron 49, 9373-9384] during the P-450cam catalyzed hydroxylation compared to 3% for the conversion of (1R)-camphor.

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The effect of pH, glycerol, temperature, and pressure on the carbon monoxide (CO) stretch mode of substrate-free cytochrome P-450cam (CYP101) was studied. Complex spectra of overlapping bands have been observed. CO stretch bands centered at about 1911-1918 cm-1 (band I), 1927-1931 cm-1 (band II), 1940-1942 cm-1 (band III), 1950-1953 cm-1 (band IV), 1960-1963 cm-1 (band V) and 1966-1973 cm-1 (band VI) are obtained from the fitting analyses independently of the lineshape model used. Only two or three bands are dominant in each spectrum. Compared to bands I, II and III, the bands IV and V are assigned to correspond to a weaker polar contact between the CO ligand and a polar group in the heme pocket (probably Thr252) because of the opposite effect of glycerol (osmotic pressure) and hydrostatic pressure on the intensity and frequency of these bands. The different CO stretch bands are interpreted as indicating conformational substates of the protein. It is suggested that water in the heme pocket plays an important role for the substate equilibrium. This substate equilibrium freezes in at the glass transition temperature, Tg, of the protein/solvent mixture. For the temperature region above Tg the thermodynamic parameters and volumes for the substates have been determined by a global fit analysis of the temperature- and pressure-dependent populations and they are compared to the respective values for camphor-bound cytochrome P-450cam.

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Pyridoxal phosphate, cofactor of several enzymes, possesses linking properties to induce oligomerization of identical proteins such as adrenodoxin-reductase or adrenodoxin. The capability to get such self-assemblies is slightly lower than that for obtaining the heterologous complex between adrenodoxin-reductase and adrenodoxin which was found to be essential for electron transfer in the cytochrome P450 system. The influence of pyridoxal phosphate on the complex formation between adrenodoxin-reductase and adrenodoxin as well as the oligomerization reaction of the isolated proteins and possible consequences is discussed.

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The effect of pH, monovalent cations, glycerol, temperature, and pressure on the carbonmonoxy (CO) stretching mode of camphor-bound cytochrome P-450cam (CYP 101) was studied. Two effects, band overlap and frequency shift, have been observed. The CO stretch infrared band located at about 1940 cm-1 is asymmetric because of the overlap of three bands at about 1931 cm-1, 1939 cm-1, and 1942 cm-1 with strongly different populations. Reducing the temperature or increasing the pressure leads to splitting the band or switching the asymmetry from the lower energy side to the higher energy side of the infrared band. The overlap of several CO stretch bands indicates conformational substates within the heme pocket. A frequency shift of the predominantly populated band is observed by changing all the parameters mentioned. The pH-induced frequency shift follows an S-shape with the pK at 6.2, which matches the pK observed for the pH-induced high-spin/low-spin transition. Conformational changes on the proximal heme side are suggested to be the origin. Monovalent cations at saturating concentration induce a small frequency shift depending on the ion radius. The potassium ion is the one that induces a CO stretch frequency with the highest wave-number while sodium and lithium (smaller radii) and rubidium and caesium ion (larger radii) have diminished values, which is supporting evidence for the special function of the potassium ion within the structure. Glycerol and hydrostatic pressure induce a red shift of the CO stretching frequency. Forced contact of the polar hydroxyl group of Thr252 of the I helix induced by pressure and indirectly by glycerol is suggested to change the CO dipole moment, reflecting in the decreased CO stretching frequency.

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The electronic absorption spectra for camphor-bound cytochrome P-450cam have been analysed in the temperature range between 78 K and 298 K. The well-known high-spin/low-spin equilibrium has been detected between 298 K and 220 K. Depending on the cooling rate, below 220 K a new species was found in the absorption spectra. In contrast, the electronic absorption spectra for camphor-free cytochrome P-450cam between 78 K and 295 K show no significant spectral changes. The conversion between the spin states of camphor-bound cytochrome P-450cam and the appearance of the new species do not correspond to the temperature-induced change in the paH value of the aqueous glycerol mixture containing phosphate or cacodylate buffer (paH 7.0). For this study a spectroscopic procedure for the determination of the temperature dependence of the paH value of the solvent for the range 78-295 K is presented using dyes as pH-indicators. It is shown that the state of the acid-base equilibrium frozen in is strongly dependent on the cooling rate of the mixture.

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The secondary structure of streptokinase (Sk) in aqueous solution was quantitatively examined by using Fourier transform infrared (FT-IR) spectroscopy. Resolution enhancement techniques, including Fourier deconvolution and derivative spectroscopy, were combined with band curve-fitting procedures to quantitate the spectral information from the amide I bands. Nine component bands were found under the broad, nearly featureless amide I bands which reflect the presence of various substructures. The relative areas of these component bands indicate an amount of beta-sheet between 30 and 37% and an alpha-helix content of only 12-13% in Sk. Further conformational substructures are assigned to turns (25-26%) and to "random" structures (15-16%). Additionally, the correlation of a pronounced component band near 1640 cm-1 (10-16% fractional area) with the possible presence of 3(10)-helices is discussed.

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Careful titration of oxidized cytochrome P-450cam from Pseudomonas putida with pyridine revealed deviations of the Eadie plot from linearity in the substrate-bound as well as in the substrate-free protein. A binding model which assumes two binding sites for pyridine--the iron and the camphor binding site--is able to describe completely the nonlinear Eadie plot.

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1. The monooxygenase and oxidase activities of liver microsomes from phenobarbital (PB)-treated rabbits were investigated for their dependence on the high spin shift (delta alpha) of the ferric cytochrome P-450 induced by a series of benzphetamine analogues. 2. The spin shift activity of the substrate determines, via the first electron transfer kinetics, the steady-state level of the reaction intermediate oxycytochrome P-450. Correlation of the amount or oxycytochrome P-450 with delta alpha can be experimentally proved. 3. The spin-state-dependent formation of oxycytochrome P-450 regulates quantitatively the rates of NADPH oxidation and substrate N-demethylation. Both activities correlate with delta alpha. Oxycytochrome P-450 is substrate-stabilized towards decay with the formation of O2- which, upon dismutation, gives rise to H2O2. 4. The ratio of N-demethylase to NADPH oxidase activity (coupling ratio) also increases with the spin shift, delta alpha. Concomitantly, the proportion of NADPH accounted for by H2O2 and H2O formation via two- and four-electron reduction of dioxygen decreases. This indicates that the substrate-induced structural changes in the enzyme active centre which give rise to spin transition may likewise modify the coupling properties. 5. Perfluorinated compounds, which fail to undergo monooxygenation, fall in line with the benzphetamine derivatives with respect to the dependence of NADPH oxidation rate and steady-state oxycytochrome P-450 level on delta alpha. The increased oxidase activity results mostly in H2O formation. 6. The leakiness of the PB-induced monooxygenase pathway in the biotransformation of oxygen in the presence of the benzphetamines and perfluorinated compounds does not result in marked increases in H2O2 formation. Therefore, the increase of NADPH oxidase activity by these substrates does not significantly enhance H2O2-mediated oxygen tissue toxicity.

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A new method for determination of the population of the high-spin state (high-spin content) in ferric cytochrome P-450 is presented. Based on curve fitting the electronic absorption spectra with a linear combination of gaussian bands analytical functions for the pure high-spin and pure low-spin states were constructed. These functions were used to fit the high-spin/low-spin mixed spectra. A good fit of the absorption spectra of six different cytochrome P-450 proteins in the presence and absence of substrates was found, indicating a similar pi-electron structure of the porphyrin and a similar chemical nature of the nearest coordination sphere of the iron in all cytochrome P-450 proteins.

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Initial reaction rates of oxygen consumption and hydrogen peroxide formation in a cytochrome P-450 catalyzed reaction are practically independent of the nature of tertiary amines that were used as substrates. From the kinetic studies and the substrate conversion results that the amount of water formed in a side reaction is determined by the substrate specificity. Both hydrogen peroxide and water formation lower the efficiency of the monooxygenatic activity of cytochrome P-450.

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