Electron transfer in hemoproteins. VIII. Influence of ionic strength on the rate of reduction of ferricytochrome c by oxymyoglobin derivatives, chemically modified at histidine residues

The influence of chemical modification of His residues in Mb on the rate of redox reaction in system MbO2--Cyt c has been studied at different ionic strengths and pH medium. The products of alkylation of all available His by bromacetate and iodacetamide, CM-Mb and CA-Mb, respectively, and myoglobin, modified by spin label 2,2', 6,6'-tetramethyl-4-bromoacetoxypiperidine-1-oxyl (SL) at His residue A10--Sl (His-A10)--Mb have been studied. It has been shown, that the character of the ionic strength dependence of reaction SL(His-A10)--MbO2 with Cyt c at pH 6.0 ann 7.0 is basically analogues to that, observed for intact protein. It means that only His-GH1 of two His residues, His-A10 and His-GH1, situated in the region of "active contact" of Mg with Cyt c molecule, participates in the interactions, essential for electron transfer. The interaction of the charge of this His with the negatively charged group of Cyt c is necessary, probably for the proper arrangement of other interactions in the active complex, because the deprotonation of His-GHl in the studied pH interval decreases the rate of the process by more than one order of magnitude. The rate of oxidation of MC-MbO2 and CA-MbO2 by ferricytochrome c, in contrast to intact protein, shows a weak dependence on the ionic strength and does not depend on the pH medium, throughout the range of ionic strengths from 0.005 to 1.0. The cause of the radical change in the ionic strength dependence is, probably, nearly entire disturbance of electrostatic interactions in the active complex due to chemical modification of His residues in the site of "active contact", and first of all, the His-CHl residue. The fact, that during alkylation of all available His in Mb the electron transfer persists in the system, points to that in the process of electron transfer to cytochrome c, uncharged group, most probably "inner" His-B5, participates. Based on the data on spatial structure and the obtained results, the positions of the charged groups in the site of "active contact" of Mb with Cyt c molecule are presented.     

Electron transport in hemoproteins. IX. The effect of zinc ions on the rate of oxymyoglobin oxidation by ferricytochrome c

The effect of zink ions, which according to the X-ray data are bound to the His GH1 residue of myoglobin, has been investigated. It is shown that the electron transfer in the system is almost completely inhibited at the equimolar Zn2+ concentration in the pH range 5 to 8. Unlike the reaction between the intact MbO2 and Cyt c, the electron transfer rate in this case does not depend on pH and ionic strength of the solution. Further increase of Zn2+ concentration up to the 20-fold molar excess has no significant effect on the rate of the process. Since the thermodynamic characteristics of the redox reaction between MbO2 and Cyt c are not altered in the presence of Zn2+, the findings obtained can be interpreted as indicating the important role of His GH1 in the formation of productive electron transfer complex.     

Electron transfer in hemoproteins. VI. The dependence of the reduction rate of ferricytochrome c by oxymyoglobin on ionic strength

The rate of the redox-reaction between MbO2 and ferri-Cyt c has been investigated in the pH range 5-8 under different ionic strength of the solution. The influence of various anions-phosphate, chloride, sulfate and acetat on the rate of the reaction were also studied. It has been shown that under the low ionic strength, I less than 0.1, all pH-dependence curves have pronounced maximum near pH 6.0. While the ionic strength values increase in this interval the reaction rate falls markedly, the profile of lg k versus square root of I/1 + square root of I is linear. Under high ionic strength values, I greater than 0.1, the reaction rate in MbO2-Cyt c system is only slightly influenced by increasing salt concentrations and by pH changing. The results obtained support the idea that the local interactions of charged groups in " active sites" of MbO2 and Cyt c play the most important role in the mechanism of electron transfer. On the contrary net charges of the molecules have a negligible effect on the rate of the reaction. Compared to anions Cl-, SO42- and CH3COO- which influence the reaction rate in an analogous way, phosphate ions have essential inhibiting effect. This is most likely explained by the specific bonding of the phosphate ions to Cty c in the immediate vicinity from the site of the "active contact" with Mb molecule.     

Mechanism of electron transfer between myoglobin derivatives and ferricytochrome C

Progress in the studies of the electron transport mechanism in biological systems is greatly hindered by the lack of detailed structural information about the components of these systems. That is why a study of electron transfer between protein molecules with the known spatial organization in model reactions in vitro is of great importance. In this respect the MbO2--Cyt C oxidation-reduction reaction offers unique possibilities. Studies of the effects of pH and ionic strength of the medium on the kinetics of this reaction in combination with chemical modification of single amino acid residues of Mb and Cyt C enabled us to identify those parts of the surface of haemoproteins where the molecules come into "active contact". A variation in the number or/and the arrangement of the charged groups at the "active sites" of the molecules induced by both changing the medium pH and chemical modification of some of these groups lowers markedly the probability of electron transfer in the system (e.g. His GH1 and His A10 in Mb) or blocks it entirely (acylation of Lys 72 (73) or Tyr 74 in Cyt C). Based on the results obtained and on the data of Mb and Cyt C X-ray analysis, the figures of spatial arrangement of the groups at the "active sites" of these molecules are presented.     

Electron transport in hemoproteins. VIII. The effect of chemical modification of ferricytochrome C on the rate of its reduction by oxymyoglobin

The influence of chemical modification of separate amino acid residues in ferricytochrome c (Cyt c) on the rate of the redox reaction with MbO2 has been studied at various pH and ionic strength values. It is shown that alkylation of His-33 and Met-65 by bromacetate does not affect the reaction rate. On the contrary, acylation of Tyr-74 or one of the neighbouring lysines, Lys-72 or Lys-73, by the spin-label N-(2,2',5,5'-tetramethyl-3-carboxypyrrolin-1-oxy)-imidazol diminishes sharply the efficiency of electron transfer in the redox system studied. Besides, unlike the reaction between native proteins, the rate of electron transfer in this case does not depend on ionic strength. The modification of Tyr-74 or Lys 72/43 does not alter the midpoint potential and the entire conformation of Cyt c. The observed effects can therefore be explained by essential disturbance of interactions, first of all, the electrostatic ones in the active complex, which is induced by the attachment of the bulky reagent to the site of "active contact" of Cyt c. Based on the obtained findings and the atomic coordinates of Cyt c, the positions of all charge and some uncharged groups on the surface of Cyt c interacting with myoglobin during electron transfer are presented.     

Catalytic effect of ferricyanide on the rate of electron transfer between myoglobin and cytochrome c]

The influence of small amounts of low-molecular electron acceptor, potassium ferricyanide, 1 to 20% relative to the cytohrome c concentration, on the rate of electron transfer in the sperm whale oxymyoglobin--horse heart cytochrome c and deoxymyoglobin--cytochrome c systems (under aerobic and anaerobic conditions, respectively) was studied. At low ionic strength, the redox reaction rate was found to increase proportionally to the concentration of ferricyanide in both redox systems. The effect depends on pH in the pH range 5-8, increasing sharply at pH < 6. It was shown that the enhancing of electron transfer is caused by the complexing of [Fe(CN)6]3- with cytohrome c in the Lys72 region, where one of the two strong binding sites for this anion is determined by NMR. Both the high ionic strength and the chemical modification of Lys72 residue inhibit this effect at low ionic strength, markedly decreasing the rate of reaction with myoglobin. Under the same conditions, the effect of ferricyanide in the reaction of oxy-Mb with yeast cytohrome c, which is isopotential to animal cytochromes c but possesses trimethylated Lys72, was several times smaller. In turn, the chemical modification of His residues in myoglobin and the complexing of zinc ion to His119(GH1) almost completely inhibit electron transfer in the systems. Thus, electron transfer between the proteins must proceed through the formation of the Mb.[Fe(CN)6]3-.Cyt c ternary complex, the contacting sites being localized in the His119(GH1) region of myoglobin and near Lys72 of cytohrome c. The increased electron transfer rate in the presence of [Fe(CN)6]3- can be explained by that its binding near Lys72, firstly, provides better electrostatic interactions in the electron transfer complex and, besides, decreases significantly (about 2-fold) the tunneling distance between the two hemes (two lengths of 1.7 and 1.2 nm instead of one of 2.9 nm).     

Controlling the electron transfer rate between oxymyoglobin and ferricytochrome C by alterations in the myoglobin contact site. The role of histidines and electrostatic and steric interactions in the mechanism of the reaction

The kinetics of the redox reaction of sperm whale and pig oxymyoglobins (MbO₂) with ferricytochrome C (CytC) from pig heart has been studied in the pH range 5–8. Also, the effects of histidine (His) modification and of the complexing of both myoglobins with Zn²⁺, on the electron transfer rate, has been investigated. It has been shown that pig MbO₂ reduces Cyt C much more effectively than sperm whale MbO₂. The pH dependence of the reaction rate is shown to result from the influence of two histidines, His 12(A10) and His 119(GH1), in the case of sperm whale myoglobin and only of His GH1 in the case of pig MbO₂. The protonation of His A10 at pH<7.5 decreases the rate of the reaction with Cyt C whereas the ionization of His GH1, on the contrary, increases the electron transfer rate 10–30 times (atI=0.03). The His residues of Cyt C are shown to have no effect on the reaction. Complexing of His GH1 with a zinc ion strongly inhibits the reaction of both sperm whale and pig MbO₂ with Cyt C. The reaction of the zinc-MbO₂ complexes, as distinct from the intact oxymyoglobins, becomes independent of pH and ionic strength. Unlike His A10, His GH1 plays a very important role in the formation of the electron transfer complexes, and is probably directly involved in the charge transfer step. Based on the data obtained, the reactive site of the Mb surface has been identified in the A-GH region. The spatial arrangement of the charged groups in the reactive sites of the two myoglobins has been obtained. The solvent accessibilities of all amino acid residues situated there have been calculated, according to Lee and Richards. In order to explain the different reactivities of sperm whale and pig myoglobins, their electrostatic properties and the steric features of the contact sites have been compared.     

The mechanism of oxymyoglobin oxidation catalyzed by ferrocyanide ions: chemically modified and mutant sperm whale myoglobins

A comparative study of the rate of ferrocyanide-catalyzed oxidation of native sperm whale MbO2, its chemically modified derivative in which all accessible His residues are alkylated by sodium bromoacetate, (CM-MbO2), and mutant sperm whale MbO2 with His119 replaced by Asp residiue, [MbO2(His119-->Asp)] was carried out. The influence of pH, ionic strength, and [Fe(CN)6]4- concentration on the oxidation rate was investigated, as well as the effect of complexing MbO2 with redox-inactive Zn2+ ion, which, at the equimolar Zn2+ concentration, forms a stable complex with His119(GH1) on the protein surface. It was shown that the mechanism of the catalysis involves specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) region, which is in agreement with a large positive electrostatic potential and the presence at this site of Mb of a cavity large enough to accommodate [Fe(CN)6]4- anion. The protonation of nearby His113 and His116 residiues (especially of the latter) plays a very important role in the catalysis, promoting the fast oxidation of bound [Fe(CN)6]4- by dissolved oxygen. Only the presence of these both necessary conditions in MbO2 structure provides its effective oxydation catalyzed by ferrocyanide.     

The oxidation of sperm whale, horse, and pig oxymyoglobins, catalyzed by ferrocyanide ions: kinetics and mechanism

The influence of pH, ionic strength of the solution, and [Fe(CN)6]4- concentration on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins, which is catalyzed by ferrocyanide ions, was studied. These myoglobins have homologous spatial structures and identical redox potentials but differ by the amount of His residues located on the protein surface. The effect of the MbO2 complexing with redox-inactive Zn2+ ion on the reaction rate was also examined. At the equimolar Zn2+ concentration, zinc ions form a stable complex with His119(GH1). It was found that the kinetic behavior of horse MbO2, which lacks His12(A10) substituted for by Gln, is fully analogous to one of sperm whale MbO2, while the oxidation of pig MbO2, three histidines of which, His12, His113(G14), and His116(G17), are replaced by Gln, is strongly inhibited. The mechanism of the catalysis was shown to involve specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) site, which is in accord with the large positive electrostatic potential of this site and the presence here of a cavity large enough to accommodate [Fe(CN)6]4-. The nearby His113 and His116 residiues, which are absent in pig Mb, also play a very important role in the catalysis, because their protonation (especially of the last residue) is most likely responsible for the week oxidation of bound [Fe(CN)6]4- by dissolved oxygen.     

Oxidation of sperm whale oxymyoglobin, catalyzed by ferrocyanide ions: kinetics and mechanisms

Specific catalytic oxidation of sperm whale oxymyoglobin by small amounts of potassium ferri- and ferrocyanide, from 1 to 20% in relation to the protein concentration, was studied. The mechanism of catalysis was shown to involve specific binding of the ferrocyanide anion to the protein. The influence of pH and ionic strength of the medium, the [Fe(CN)6]4- concentration and of chemical modification of Mb histidines by bromoacetate, as well as the effect of the Mb complexing with redox-inactive zinc ion on the rate of reaction was examined. The zinc ion forms a stable complex with His 119(GH1) on the Mb surface at the equimolar Zn2+ concentration. The kinetic scheme of the reaction was analyzed, and the equilibrium and kinetic parameters were obtained. It was first shown that the strong oxidant such as potassium ferricyanide is able to react with the same protein by two distinct mechanisms: (i) a simple outer sphere electron transfer over the heme edge and (ii) electron transfer after the specific binding of [Fe(CN)6]4- to oxyMb in the His 119(GH1) region, thus catalyzing the protein oxidation.     

Catalytic effect of ferricyanide between myoglobin and luminol and effect of temperature.

Specific catalytic oxidation of oxymyoglobin (MbO(2)) and luminol by ferricyanide was studied in a flow-injection system. MbO(2) in different redox states (ferric and ferrous) was oxidized to Mb(Fe(III)) by ferricyanide, and then specific binding of the ferrocyanide anion to Mb(Fe(III)) to the His 119 (GH1) region accelerated the electron transfer between Mb(Fe(III)) and luminol, which produced a chemiluminescence (CL) signal at 425 nm. The increased CL emission was correlated with the myoglobin concentration in the range 0.16-7.5 microg/mL. Thermogravimetry and differential scanning calorimetry were used to investigate the temperature effects on this reaction. The results showed that the CL intensity in the presence of myoglobin changed considerably with heating in the range 15-50 degrees C, and the maximal CL intensity was observed at 40 degrees C, corresponding to the glass transition temperature of myoglobin. The effect of different ligands and interferences were also studied.     

Electron transfer between liposomal cytochrome c1 and cytochrome c: catalytic implications of electrostatic potentials

Kinetics measurements of the electron transfer between ferricytochrome c and liposomal ferrocytochrome c1 (with and without the hinge protein) were performed. The observed rate constants(kobs) of electron transfer between liposomal ferrocytochrome c1 and ferricytochrome c at different ionic strengths were measured in cacodylate buffer, pH 7.4, at 2 C. The effect of ionic strength on the rate constant(kobs) of electron transfer between liposomal cytochrome c1 and cytochrome c is far greater than that in the solution kinetics (Kim, C.H., Balny, C. and King, T.E. (1987) J. Biol. Chem. 262, 8103-8108). The result demonstrates that the membrane bound cytochrome c1 creates a polyelectrolytic microenvironment which appears to be involved in the control of electron transfer and can be modulated by the ionic strength. The involvement of electrostatic potentials in the electron transfer between the membrane bound cytochrome c1 and cytochrome c is discussed in accord with the experimental results and a polyelectrolyte theory.     

Mapping the electron transfer interface between cytochrome b5 and cytochrome c

To characterize the cytochrome b(5) (Cyt b(5))-cytochrome c (Cyt c) interactions during electron transfer, variants of Cyt b(5) have been employed to assess the contributions of electrostatic interactions (substitution of surface charged residues Glu44, Glu48, Glu56, and Asp60 and heme propionate), hydrophobic interactions, and the thermodynamic driving forces (substitutions for hydrophobic residues in heme pocket residues Phe35, Pro40, Val45, Phe58, and Val61). The electrostatic interactions play an important role in maintaining the stability and specificity of the Cyt b(5)-Cyt c complex that is formed. There is no essential effect on the intraprotein complex electron transfer even if most of the involved negatively charged residues on the surface of Cyt b(5) have been removed. The results support a dynamic docking paradigm for Cyt b(5)-Cyt c interactions. The orientation that is optimal for binding may not be optimal form for electron transfer. Substitution of hydrophobic residues does not have a significant effect on the binding between Cyt b(5) and Cyt c; rather, it regulates the electron transfer rates via changes in the driving force. Combining the electron transfer studies of the Cyt b(5)-Cyt c system and the Cyt b(5)-Zn-Cyt c system, we obtain the reorganization energy (0.6 eV) at an ionic strength of 150 mM.     

Ionic strength dependence of the non-physiological electron transfer between flavodoxin and cytochrome c 553 from D. vulgaris

A hypothetical model for the non-physiological electron transfer complex between cytochrome c553 (c553) and the flavodoxin (fld) from the sulphate-reducing bacteria Desulfovibrio vulgaris has been recently published [1] based on rigid-body docking and refined by molecular dynamics. In this study, the functional validity of this model is tested by looking at the role of electrostatics in the non-physiological interprotein electron transfer between the two proteins at different ionic strengths. The results are compared with the electron transfer between fld and cytochrome c from horse heart (hhc). Second-order rate constants (k2) were measured for both non-physiological systems at different ionic strengths: a complex, bell-shaped behaviour is observed for the k2 of the c553/fld redox pair with an optimum rate at I=58 mmol l(-1), whereas under the same conditions the k2 for hhc/fld decreased monotonically with increasing ionic strength. Results from the electron transfer kinetics are rationalised in terms of reorganisational effects of an ensemble of conformations of the electron transfer competent c553/fld complexes, consistent with the published model.     

Site-directed mutagenesis of yeast cytochrome c peroxidase shows histidine 181 is not required for oxidation of ferrocytochrome c

The long-distance electron transfer observed in the complex formed between ferrocytochrome c and compound I, the peroxide-oxidized form of cytochrome c peroxidase (CCP), has been proposed to occur through the participation of His 181 of CCP and Phe 87 of yeast iso-1 cytochrome c [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330]. We have examined the role of His 181 of CCP in this process through characterization of a mutant CCP in which His 181 has been replaced by glycine through site-directed mutagenesis. Data from single-crystal X-ray diffraction studies, as well as the visible spectra of the mutant CCP and its 2-equiv oxidation product, compound I, show that at pH 6.0 the protein is not dramatically altered by the His 181----Gly mutation. The rate of peroxide-dependent oxidation of ferrocytochrome c by the mutant CCP is reduced only 2-fold relative to that of the parental CCP, under steady-state conditions. Transient kinetic measurements of the intracomplex electron transfer rate from ferrous cytochrome c to compound I indicate that the rate of electron transfer within the transiently formed complex at high ionic strength (mu = 114 mM, pH = 6) is also reduced by approximately 2-fold in the mutant CCP protein. The relatively minor effect of the loss of the imidazole side chain at position 181 on the kinetics of electron transfer in the CCP-cytochrome c complex precludes an obligatory participation of His 181 in electron transfer from ferrous cytochrome c to compound I.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reaction mechanism of Photosystem I reduction by plastocyanin and cytochrome c6 follows two different kinetic models in the cyanobacterium Pseudanabaena sp. PCC 6903

Plastocyanin (Pc) and cytochrome c₆ (Cyt) have been purified to homogeneity from the cyanobacterium Pseudanabaena sp. PCC 6903, which occupies a unique divergent branch in the evolutionary tree of oxygen-evolving photosynthetic organisms. The two metalloproteins have similar molecular masses (9–10 kDa), as well as almost identical isoelectric points (ca. 8) and midpoint redox potentials (ca. 350 mV, at pH 7). Their reaction mechanism of electron transfer to Photosystem I (PS I) has been analyzed by laser-flash absorption spectroscopy. The kinetic traces with Pc correspond to monophasic kinetics, whereas those with Cyt are better fitted to biphasic curves. The observed pseudo first-order rate constant (k_obs) with Pc and that for the slower phase with Cyt exhibit saturation profiles at increasing donor protein concentrations, thereby suggesting that the two metalloproteins are able to form transient complexes with PS I. The ionic strength dependence of the rate constants for complex formation makes evident the electrostatic nature of intermediate complexes. The experimental findings indicate that the PS I reduction kinetics in Pseudanabaena follow a type II mechanism with Pc and a type III mechanism with Cyt, according to the different kinetic models proposed previously [(Hervás M, Navarro JA, Díaz A, Bottin H and De la Rosa MA (1995) Biochemistry 34: 11321–11326)]. From an evolutionary point of view, this reinforces our previous observation that PS I was first adapted to operate efficiently with positively charged Cyt rather than with Pc.     

The reaction of cytochrome aa3 with (porphyrin) cytochrome c as studied by pulse radiolysis

(1) Using the pulse-radiolysis and stopped-flow techniques, the reactions of iron-free (porphyrin) cytochrome c and native cytochrome c with cytochrome aa3 were investigated. The porphyrin cytochrome c anion radical (generated by reduction of porphyrin cytochrome c by the hydrated electron) can transfer its electron to cytochrome aa3. The bimolecular rate constant for this reaction is 2 x 10(7) M-1 . s-1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20 degrees C). (2) The ionic strength dependence of the cytochrome c-cytochrome aa3 interaction was measured in the ionic strength range between 40 and 120 mM. At ionic strengths below 30 mM, a cytochrome c-cytochrome aa3 complex is formed in which cytochrome c is no longer reducible by the hydrated electron. A method is described by which the contributions of electrostatic forces to the reaction rate can be determined. (3) Using the stopped-flow technique, the effect of the dielectric constant (epsilon) of the reaction medium on the reaction of cytochrome C with cytochrome aa3 was investigated. With increasing epsilon the second-order rate constant decreased.     

Kinetics of intracomplex electron transfer and of reduction of the components of covalent and noncovalent complexes of cytochrome c and cytochrome c peroxidase by free flavin semiquinones

The kinetics of reduction of free flavin semiquinones of the individual components of 1:1 covalent and electrostatic complexes of yeast ferric and ferryl cytochrome c peroxidase and ferric horse cytochrome c have been studied. Covalent cross-linking between the peroxidase and cytochrome c at low ionic strength results in a complex that has kinetic properties both similar to and different from those of the electrostatic complex. Whereas the cytochrome c heme exposure to exogenous reductants is similar in both complexes, the apparent electrostatic environment near the cytochrome c heme edge is markedly different. In the electrostatic complex, a net positive charge is present, whereas in the covalent complex, an essentially neutral electrostatic charge is found. Intracomplex electron transfer within the two complexes is also different. For the covalent complex, electron transfer from ferrous cytochrome c to the ferryl peroxidase has a rate constant of 1560 s-1, which is invariant with respect to changes in the ionic strength. The rate constant for intracomplex electron transfer within the electrostatic complex is highly ionic strength dependent. At mu = 8 mM a value of 750 s-1 has been obtained [Hazzard, J. T., Poulos, T. L., & Tollin, G. (1987) Biochemistry 26, 2836-2848], whereas at mu = 30 mM the value is 3300 s-1. This ionic strength dependency for the electrostatic complex has been interpreted in terms of the rearrangement of the two proteins comprising the complex to a more favorable orientation for electron transfer. In the case of the covalent complex, such reorientation is apparently impeded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of electron transfer between cytochrome c and laccase.

Rate constants have been determined for the electron-transfer reactions between reduced horse heart cytochrome c and resting Rhus vernicifera laccase as a function of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of reduced cytochrome c was determined to be k = 125 M-1 s-1 at 25 degrees C in 0.2 M phosphate buffer at pH 6.0 with the activation parameters delta H++ = 16.2 kJ mol-1 and delta S++ = 28.9 J mol-1 K-1. The rate constants increased with decreasing buffer concentration, indicating that electron transfer from cytochrome c to laccase is favored by the local electrostatic interaction (ZAZB = -0.9 at pH 6 and -1.3 at pH 4.8) between the basic proteins with positive net charges. From the increase of the rate of electron transfer with decreasing pH, one of the driving forces of the reaction was suggested to be the difference in the redox potentials between the type 1 copper in laccase and the central iron in cytochrome c. Further, on addition of one hexametaphosphate anion per cytochrome c molecule, the rate of the electron transfer was increased, probably because the association of both proteins became more favorable.     

Effect of electrostatic interactions on the percolation concentration of fibrillar beta-lactoglobulin gels

The effect of electrostatic interactions on the critical percolation concentration (c(p)) of fibrillar beta-lactoglobulin gels at pH 2 was investigated using rheological measurements, transmission electron microscopy (TEM), and performing conversion experiments. A decreasing c(p) with increasing ionic strength was found. The fraction of nonaggregated beta-lactoglobulin was independent of ionic strength in the regime of 0.01-0.08 M. TEM experiments showed long fibrils (2-7 microm) for ionic strengths between 0.01 and 0.08 M. Since both the conversion of monomers and the contour length of the fibrils were independent of ionic strength (0.01-0.08M), the linear increase of c(p) with the Debye length can be attributed purely to an increase of electrostatic repulsion between the fibrils. This increase is explained in terms of an adjusted random contact model which takes into account the charge and semiflexibility of the fibrils.