Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrateferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the K_m (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the ‘association’ rate but also by increasing the ‘dissociation’ rate for bound cytochrome c converting the ‘primary’ (T) site at high salt concentrations into a site similar kinetically to the ‘secondary’ (L) site at low ionic strength. A finite K_m of 170 μM at very high ionic strength indicates a ratio of $\text{K}{}^{\mathrm{\infty }}{}_{\mathrm{<mtext>M</mtext>}}\text{K}{}^0{}_{\mathrm{<mtext>M</mtext>}}$ of about 5000. It is proposed that anions either modify the E¹₀ of cytochrome c bound at the primary (T) site or that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

Slow,Reversible, Coupled Folding and Binding of the Spectrin Tetramerization Domain

Many intrinsically disordered proteins (IDPs) are significantly unstructured under physiological conditions. A number of these IDPs have been shown to undergo coupled folding and binding reactions whereby they can gain structure upon association with an appropriate partner protein. In general, these systems display weaker binding affinities than do systems with association between completely structured domains, with micromolar K_d values appearing typical. One such system is the association between α- and β-spectrin, where two partially structured, incomplete domains associate to form a fully structured, three-helix bundle, the spectrin tetramerization domain. Here, we use this model system to demonstrate a method for fitting association and dissociation kinetic traces where, using typical biophysical concentrations, the association reactions are expected to be highly reversible. We elucidate the unusually slow, two-state kinetics of spectrin assembly in solution. The advantages of studying kinetics in this regime include the potential for gaining equilibrium constants as well as rate constants, and for performing experiments with low protein concentrations. We suggest that this approach would be particularly appropriate for high-throughput mutational analysis of two-state reversible binding processes.     

Functional binding of cardiolipin to cytochromec oxidase

Bovine cytochromec oxidase usually contains 3–4 mol of tightly bound cardiolipin per cytochromeaa ₃ complex. At least two of these cardiolipins are required for full electron transport activity. Without the tightly bound cardiolipin, cytochromec oxidase has only 40–50% of its original activity when assayed in detergents that support activity, e.g., dodecyl maltoside. By measuring the restoration of electron transport activity, functional binding constants for cardiolipin and a number of cardiolipin analogues have been evaluated (K _d,app=1 µM for cardiolipin). These binding constants agree reasonably well with direct measurement of the binding using [¹⁴C]-acetyl-cardiolipin (K _d <0.1 µM) when the enzyme is solubilized with Triton X-100. These data are discussed in relationship to the wealth of data that is known about the association of cardiolipin with cytochromec oxidase and the other mitochrondrial electron transport complexes and transporters.     

In vitro characterization of skeletal muscle β-adrenergic receptors coupled to adenylate cyclase

[³H]Dihydroalprenolol, a potent ß-adrenergic antagonist, was used to identify the adenylate cyclase-coupled ß-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed [³H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [³H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [³H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions.The binding of [³H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K₁) and dissociation (K₂) rate constants of 2.21 · M^? · min^?1 and 3.21 · 10^?1, respectively, were obtained. The dissociation constant (K_d) of 15 nM for [³H]dihydroalprenolol obtained from saturation binding data closely agreed with the (K_d) derived from the ratio of dissociation and association rate constants (K₂/K₁).Several β-adrenergic agents known to be active on intact skeletal muscle also competed for [³H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [³H]dihydroalprenolol binding sites with a potency of isoproterenol > epinephrine > norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (?)-isomers being more than potent than (+)-isomers. Phenylephrine, an α-adrenergic agonist, showed no effect either on [³H]dihydroalprenolol binding or on adenylate cyclase. Known ß-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [³H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The (K_i) values for the antagonists determined from inhibition of [³H]dihydroalprenolol binding agreed closely with the (K_i) values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [³H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the ß-adrenergic receptor.     

Nature of the individual Ca2+ binding sites in Ca2+-regenerated bacteriorhodopsin

The binding constants, K₁ and K₂, and the number of Ca²⁺ ions in each of the two high affinity sites of Ca²⁺-regenerated bacteriorhodopsin (bR) are determined potentiometrically at different pH values in the range of pH 3.5-4.5 by using the Scatchard plot method. From the pH dependence of K₁ and K₂, it was found that two hydrogen ions are released for each Ca²⁺ bound to each of the two high affinity sites. Furthermore, we have measured by a direct spectroscopic method the association constant, K_s, for the binding of Ca²⁺ to deionized bR, which is responsible for producing the blue to purple color change. Comparing the value of K_s and its pH dependence with those of K₁ and K₂ showed that the site corresponding to K_s is to be identified with that of K₂. This is in agreement with the conclusion reached previously, using a different approach, which showed that it is the second Ca²⁺ that causes the blue to purple color change.

Our studies also show that in addition to the two distinct high affinity sites, there are about four to six sites with lower binding constants. These are attributed to the nonspecific binding in bR.

     

Association Constants for Metal Hexacyanide Binding to Cytochrome c

The binding of[Co(CN)₆]^3?, and that of[Fe(CN)₆]^3? and [Ru(CN)₆]^4? using a competitive method, to horse cytochrome c has been studied by ⁵⁹ Co NMR spectroscopy. At I = 0.07 M, without added salt and in ²H₂O at ph* 7.3 (measured in ²H₂O) and 25°C, there are at least two binding sites on ferricytochrome c and ferrocytochrome c for [Co(CN)₆]^3?. Association constants were determined to be 2.0 ± 0.6 × 10³M^?1 and 1.5 ± 0.5 × 10²M^?1 respectively. with no effect of the oxidation state of the cytochrome. At higher ionic strength (I = 0.12 M adjusted with KCl the binding markedly decreased, and, although it was not possible to determine the precise binding stoichiometry and magnitude of association constants, it is clear that the association constants are ≤ 1.5 × 10^tM^?1 The binding of [Ru(CN)₆]^4? at I = 0.07, without added salt and in ²H₂O at pH 1.3 and 23°C, was not precisely defined, but its binding strength relative to that of [Fe(CN)₆]^3? was determined. Extrapolating this to I = 0.12 (KCl) suggests that under these conditions the association constant for [Ru(CN)₆]^4? binding to ferricytochrome c is ≤ 3 × 10²M^?1.     

The binding of Arg- and Lys-peptides to single standed polyribonucleotides and its effect on the polymer conformation

The interactions between basic oligopeptides (Lys₂, Lys₃, Arg₂, and Arg₃) and single stranded polynucleotides (poly(A), poly(C), poly(I) and poly(U) were investigated at low ion concentration by UV spectroscopy, circular dichroism and field jump relaxation. Various domains of binding were detected: 1) High concentrations (up to 1 mM) of some peptides induce opalescencs followed by coacervation- Arg₃ causes coacervation in all polynucleotides used, yet Lys₃ only in poly(I). In the case of poly(I) the threshold concentration for coacervation is much lower for Arg₃ (150 μM) than for Lys₃ (500 μM). 2) Medium concentrations (?10 μM) of Arg₃ and Lys₃ induce helix formation in poly(U). In the case of poly(I) cooperative helix formation is only induced by Lys₃, but not by Arg₃. 3) The onset of peptide association is observed at very low peptide concentrations (?1 μM) already by using the field jump method. The association is reflected by a relaxation process, that can be described by a single exponential within experimental accuracy. Measurements of relaxation time constants as a function of the peptide concentration provide information on the association constants K, the number of nucleotide residues per binding place n and the rate constants k_R and k_D. Using a simple model with independent and “separate” binding sites, K for Arg₃ and Lys₃ is found to be in the range of 10⁶ to 10⁷ M^?1. In the case of Arg₂ and K is lower by a factor of about 10. For various polynucleotides K_Arg3 is slightly higher than K_Lys3. except in the case of poly(I), where K_Arg3/K_Lys3 ≈ 5. Similar data are obtained by application of a “sphere model” (see below). These results provide quantitative evidence for specific hydrogen bonding between the guanidino group of Arg and inosine. They also explain the absence of helix formation for poly(I) + Arg₃: Arg blocks the hydrogen bonding sites of inosine. Thus cooperative coupling leads in this case to a considerable amplification of specificity in the peptide-polynucleotide interaction Both field jump and stopped flow data demonstrate a high mobility of the peptide lisands along the polymer, resulting in a redistribution being fast compared with the overall binding step. Based on this result the relaxation data are analysed by a “sphere” model, which considers a) excluded binding under the condition of fast Ugand distribution along the lattice and b) the connection of sites into a polymer sphere. The rate constants obtained by this model are in the range of 4 × 10¹¹ M^?1 s^?1. These high values reflect the large reaction distance for polymers of chain lengths around 1000. A comparison with rate constants obtained previously for oligomer complexes indicates that the recombination rate is approximately a function of the square root of the nucleotide chain length, which is directly related to the mean radius of coiled polymers.     

NADPH-cytochrome c reductase in outer membrane of kidney mitochondria. Purification and properties.

NADPH-cytochrome c reductase of vitamin D₃-deficient chick kidney mitochondria has been purified approximately 1100-fold to a specific activity of 788 nmol cytochrome c reduced/min/mg protein. Analytical gel electrophoresis of the purified enzyme revealed two bands when stained for protein, but only the more anodic band demonstrated NADPH-tetrazolium reductase activity. The relative ease of solubilization of the reductase without the use of lipases, proteases, or detergents was the first line of evidence that suggested a novel mitochondrial localization for this previously unreported NADPH-linked cytochrome c reductase. The apparent properties of the reductase suggest that the enzyme is a component of kidney mitochondrial outer membrane. The kinetic determination of Michaelis constants with respect to NADPH, cytochrome c, and NADH gave the following values: K_mNADPH = 1.7 μM, K_mcytc = 3.4 μM, and K_mNADH = 20 mM. These constants were different from those of the intact kidney microsomal reductase: K_mNADPH = 5.5 μM, K_mcytc = 10.5 μM, and K_mNADH = 13.3 μM. The mitochondrial as well as the intact microsomal reductases exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction. Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN. The oxidized enzyme has absorption maxima at 280 and 450 nm with a shoulder at 415 nm. Upon reduction with NADPH a distinct loss in the 450-nm absorption was observed. Ouchterlony immunodiffusion studies with rabbit antiserum to chick renal mitochondrial ferredoxin did not reveal cross-reactivity when the purified reductase was the antigen. This excludes the involvement of a ferredoxin-type iron-sulfur protein in the NADPH-mediated reduction of cytochrome c by the purified reductase.     

Absence of multiple forms of ribulose 15-bisphosphate carboxylase/oxygenase in mung bean

Ribulose 1,5-bisphosphate carboxylase/oxygenase has been reported to occur in multiple forms in mung bean (Phaseolus aureus) using Sephadex G-200 chromatography. We have isolated this enzyme by identical methodology. The profile from Sephadex G-200 chromatography shows only one peak in contrast to the previous report and we find no evidence to corroborate the conclusions. Where V_c, V_o and K_c, K_o represent V_max and Michaelis constants, respectively, the constant V_cK_o/V_oK_c for the single form is 70 at 40 μM CO₂ and 1200 μM O₂.     

Purification and characterization of the cytochrome P-448 component of a benzo(a)pyrene hydroxylase from Saccharomyces cerevisiae

Cytochrome P-448, a type of cytochrome P-450, from brewer's yeast (Saccharomyces cerevisiae) grown under conditions of glucose repression was isolated and purified. Triton X-100 in very low concentration proved to be very effective in stabilizing P-448 in the microsomal fraction and later prevented its conversion to cytochrome P-420 through solubilization with various ionic and nonionic detergents. Highest yields were obtained with 1% sodium cholate, in the presence of 0.1% Triton X-100 and reduced glutathione. A novel combination of hydrophobic adsorption and other chromatographic techniques was used for the purification of cytochrome P-448. These involve the use of amino octyl-Sepharose 4B, instead of the low-yielding aminohexyl derivative, followed by the fast-running hydroxyapatite-cellulose column. Finally, the use of DEAE-Sephacel was found to increase greatly the purity of the cytochrome P-448 obtained. The molecular weight of this preparation was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (M_r, 55,500). Using the known molar extinction coefficient of the carbon monoxide-difference spectrum the estimate of degree of purity of cytochrome P-448 obtained by this purification procedure was between 88 and 97%. Electrophoresis also showed that this preparation was completely homogeneous and assays showed that it was also completely free of cytochrome b_s, cytochrome c reductase and cytochrome P-420. Purified cytochrome P-448 reconstituted with cytochrome P-450 (cytochrome c) reductase, isolated from yeast, showed 10-fold higher aryl hydrocarbon hydroxylase activity with benzo[a]pyrene as a substrate than the corresponding microsomal fraction enzyme. Kinetics of benzo[a]pyrene hydroxylation were determined: K_m (33 μm) was comparable with that reported for purified hepatic cytochrome P-448. The number of binding sites of microsomal and purified cytochromes P-450 (from liver of phenobarbital-induced rats) and yeast cytochrome P-448 with benzo[a]pyrene has been determined using and equilibrium gel filtration method. There is one binding site in each case (contrast with six sites for microsomal enzymes). The Scatchard plot gives number of binding sites, apparent association constants (K), and the equivalent dissociation constants (K_s). Comparison is made with spectral dissociation constants for these enzymes and benzo[a]pyrene. Thus the proportion bound, dissociation constant (K_s), and stoichiometry of rat liver (phenobarbital induced) and yeast cytochrome P-448 with benzo[a]pyrene were compared with corresponding values for microsomal fractions of both systems. Purified enzymes had higher K_s values in both cases, and the proportion of enzyme that bound benzo[a]pyrene was high (53%) for liver and this value is 100% for purified enzyme from yeast, which is the same as the value obtained for the microsomal enzyme from yeast.     

Analysis of ligand binding curves in terms of species fractions

The ligand binding curve for a macromolecular system presents the average number or ligand molecules bound per macromolecule as a function of the chemical potential or the logarithm of the ligand concentration. We show that various observable properties of this curve, for example its asymptotes and derivatives, are expressible in terms of linear combinations of the mole fractions α_i of macromolecules binding i molecules of ligand. Whenever enough such properties of the binding curve are known, the linear equations in α_i can be solved to give the mole fractions of each of the various macromolecular species. An application of these results is that a Hill plot for hemoglobin-ligand equilibrium where the asymptotes approach unit slope can be made to yield the four Adair constants by a simple algebraic method. A second use is that a knowledge of the first and second derivatives of the binding curve at points along the curve can yield the species fractions as functions of the degree of saturation without direct knowledge of the ligand binding constants. These methods are illustrated by some numerical examples.     

Muscarinic receptor sites in human foetal brain

The specific binding of the muscarinic cholinergic ligand N-methylscopolamine to human foetal brain has been measured. A level of binding of 64 pmol/g protein was found with a dissociation constant, K_d of 0.27 nM. Values of 0.17 nM min^?1 and 0.048 min^?1 for the association rate constant, K_on, and the dissociation rate constant K_off respectively, were obtained. The pharmacological properties of the binding site were found to be very similar to those reported for muscarinic receptors from adult mammalian brain except that the binding of pirenzepine and the nicotinic antagonists d-tubocurarine and decamethonium shows differences from that seen in adult brain.     

Kinetic characterization of small DNA-binding molecules interacting with a DNA strand on a quartz crystal microbalance

Quantitative studies of the binding of various DNA-binding antibiotics with dsDNA are useful for drug design, not only for effective antibiotics, but also for antitumor drugs. We studied the binding kinetics, association and dissociation rate constants, and association constants (k_on, k_off, and K_a, respectively) of intercalators and groove binders, including various antibiotics, to double-stranded DNA (dA₃₀·dT₃₀ and dG₃₀·dC₃₀) immobilized on a highly sensitive 27 MHz quartz-crystal microbalance (QCM) in aqueous solution. Although a simple ethidium bromide intercalator bound to both dA₃₀·dT₃₀ and dG₃₀·dC₃₀, antibiotics that are side-binding intercalators, such as daunomycin, aclacinomycin A, and actinomycin D, with sugar or peptide moieties on the intercalator parts selectively bound to dG₃₀·dC₃₀ with high K_a and small k_off values. Nogalamycin, a dumbbell-shaped penetrating intercalator, showed low k_on and k_off values owing to slow duplex unwinding during the penetration process. Groove binders (Hoechst 33258, distamycin A, and mithramycin) had high K_a values owing to the high k_on values. Kinetic parameters depended largely on molecular shapes and DNA-binding molecule binding modes.     

Interpolation method for accurate affinity ranking of arrayed ligand–analyte interactions

The values of the affinity constants (k_d, k_a, and K_D) that are determined by label-free interaction analysis methods are affected by the ligand density. This article outlines a surface plasmon resonance (SPR) imaging method that yields high-throughput globally fitted affinity ranking values using a 96-plex array. A kinetic titration experiment without a regeneration step has been applied for various coupled antibodies binding to a single antigen. Globally fitted rate (k_d and k_a) and dissociation equilibrium (K_D) constants for various ligand densities and analyte concentrations are exponentially interpolated to the K_D at R_max = 100 RU response level (K_D^R100).     

Functional and structural analyses on abnormal hemoglobins with impaired oxygen binding properties—To elucidate the allosteric mechanism of hemoglobin

The altered oxygen binding curves for various abnormal hemoglobins were analyzed according to a two-state allosteric model. Of three allosteric parameters computed for abnormal hemoglobins, K _R was nearly constant, but K _T and L varied with the correlation of log c=?0.4 log L, where c is K _R/K _T. This correlation indicates that the abnormal allosteric oxygen binding of hemoglobin is due to altered molecular properties of the deoxy-T state but not that of the deoxy-R state. To clarify the molecular basis of this idea, resonance Raman spectra in the low-frequency region of abnormal hemoglobins were measured under different solvent conditions. Varied frequencies of iron-histidine stretching Raman lines was found to correlate with varied oxygen affinities (K _T) of deoxy-T states. The strength of the iron-histidine bond of deoxy-T states was changed, depending upon the magnitude of the strain imposed on hemes by globin, and this bond presumably comprises an important part of the regulation mechanisms for hemoglobin oxygen binding and structure changes.     

Intracellular Potassium : A Determinant of the Sodium-Potassium Pump Rate

Normal human red cells which have had their intracellular sodium (Na_c) reduced have a diminished Na-K pump rate, but only if intracellular potassium (K_c) is high. If most of the K_c is replaced by tetramethylammonium or choline, both ouabain-sensitive Na efflux and K influx are significantly increased even with Na_c below normal. Cells with reduced Na_c and high K_c have an unchanged Na efflux if external potassium (K_ext) is removed. In contrast, low-Na, low-K cells have a large ouabain-sensitive Na efflux which shows a normal response to removal of K_ext. Neither low-K nor high-K cells have an altered ouabain-sensitive K efflux. Measurement at constant low Na_c and varying K_c shows the pump Na efflux to be an inverse function of K_c. Thus, in low-Na cells, K_c appears to act as an inhibitor of the pump. Inhibition by high K_c can be seen even when Na_c is normal. The effects attributed to K_c are distinguished experimentally from other variables such as cell volume, adenosine triphosphate concentration, effects of the replacement cations, and the method used to alter intracellular cation concentrations. A role is proposed for K_c, in cooperation with Na_c, in regulating the pump rate of normal human red cells.     

Nucleotides do not modulate rat luteocyte human chorionic gonadotropin responsiveness by inhibiting human chorionic gonadotropin binding

Nucleotide inhibition of ¹²⁵I-labeled human chorionic gonadotropin binding to luteocyte receptor was studied by investigating effects of nucleotides on the apparent equilibrium association constant (K_a) and number of binding sites (B_max), and on rate constants for association (k₊₁) and dissociation (k_?1, k_?2). K_aandB_max were determined by various analyses of equilibrium binding data using washed 2000g pellet of an ovarian homogenate from rats 7 days after pregnant mare's serum gonadotropin-human chorionic gonadotropin priming. Adenyl and guanyl nucleotides, as well as other nucleotides, lowered the K_a of ¹²⁵I-labeled human chorionic gonadotropin binding to luteocyte receptor without affecting B_max. The degree of inhibition was dose related at nucleotide concentrations greater than 10^?3 m. GTP and guanyl-5′-ylimidodiphosphate inhibitions were similar in the presence or absence of EDTA (1.25 × 10^?3 m). ATP and GTP lowered K_a by slowing the rate of association. Inhibition of binding could not be demonstrated at lower nucleotide concentrations even when luteocyte membranes were purified partially by sucrose density gradient ultracentrifugation. In light of the high nucleotide concentrations required to inhibit ¹²⁵I-labeled human chorionic gonadotropin binding and the inhibition by Mg²⁺ and PP₁ at similar concentrations, the effect appears to be a nonspecific ionic effect. Therefore, in contrast to the glucagon-hepatocyte system, luteocyte human chorionic gonadotropin responsiveness does not appear to be modulated by nucleotide inhibition of human chorionic gonadotropin-receptor interaction.     

The interaction of nucleotides and yeast hexokinase

The kinetics of ethenoadenosine triphosphate (?ATP) as the phosphate donor in the phosphoryl transfer reaction of hexokinase were examined to obtain the K_m′s, V's, and K_α's for the nucleotide and sugar. Dissociation constants for eATP and ?ADP with hexokinase were obtained from fluorometric measurements and compared with similar constants obtained kinetically. Other selected nucleoside triphosphates were used as phosphate donors in the hexokinase reaction and their kinetic constants were obtained. Reactions were also performed using two nucleotides simultaneously as phosphorylating substrates for the hexokinase reaction in an attempt to find the individual dissociation constants, K_m′s and K_i′s. These were compared with the K_m′s obtained from using the nucleotides separately in the hexokinase reaction. From these kinetic and fluorescence binding studies, evidence is presented supporting the postulate that the K_m′s are primarily dissociation constants in a random bi-bi mechanism. Analysis of the K_m values provides additional evidence to support the importance of the amino group in position 6 on the purine ring as a hydrogen-bond acceptor during binding. It was found that ?CTP was a much better hexokinase substrate than CTP. These observations suggest that the V for this reaction is highly dependent upon the size of the nucleotide.     

Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Several cytochromes c₂ from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (pK_o) and one in the reduced form (pK_r). These cytochromes fall into one of two groups according to the degree of separation of pK_o and pK_r. In group A, represented here by the Rhodomicrobium vannielii cytochrome c₂, the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c₂ and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the pK of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised pK for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c₂, the separation between pK_o and pK_r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c₂/mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c-557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c₂. Using Coulomb's law, it was found that the energy required to separate pK_o and pK_r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.     

Possibilitiés d'étude des variantes de la cholinestérase plasmatique humaine par électrophorèse d'affinitéPossibilities of study of the human plasma cholinesterase variants by affinity electrophoresis

Affinity electrophoresis has been applied to the study of the multiple molecular forms of three human plasma cholinesterase phenotypes (usual enzyme U, atypical enzyme A and intermediate UA). Electrophoreses were carried out in polyacrylamide gels containing a water-soluble macromolecular derivative of m-amino-(substituted)-phenyltrimethylammonium immobilized within the gel network.Apparent dissociation constants (K_{D app}) were estimated from the mobilities of the enzymes versus ligand concentration.The ratio of K_{D app} values of the molecular forms of phenotypes A and U which is approximately 2 is consistent with the hypothesis that the anionic site is altered in atypical enzyme.