Characterization of the polyanion-induced molten globule-like state of cytochrome c

Cytochrome c (cyt c) undergoes a poly(vinylsulphate) (PVS)-induced transition at slightly acidic pH into a molten globule-like state that resembles the effect that negatively charged membrane surfaces have on this protein. In this work, the thermodynamic properties of the molten globule-like state of cyt c in complex with PVS are studied using differential scanning calorimetry, circular dichroism, fluorescence, and absorbance spectroscopy. The temperature-induced transition of the molten globule-like state of cyt c in the complex with PVS is characterized by a significantly lower calorimetric enthalpy than in the "typical" molten globule state of cyt c, i.e. free protein at pH 2.0 in high ionic strength. Moreover, the thermally-denatured state of cyt c in the complex at pH < 6 contains nearly 50% of the native secondary structure. The dependence of the transition temperature on the pH indicates a role for histidine residues in the destabilization of the cyt c structure in the PVS complex and in stabilization of the denatured state with the residual secondary structure. A comparison of the effects of small anions and polyanions demonstrates the importance of cooperativity among the anions in the destabilization of cyt c. Predictably, other hydrophilic flexible polyanions such as heparin, polyglutamate, and polyadenylate also have a destabilizing effect on the structure of cyt c. However, a correlation between the properties of the polyanions and their effect on the protein stability is still unclear.     

Preparation of polyaniline-modified electrodes containing sulfonated polyelectrolytes using layer-by-layer techniques

Polyaniline (PAni) has been used frequently for the construction of biosensors. However, a prime limitation is its instability at basic or neutral pH because of the loss of its electrochemical activity and conductivity. In this study, three available sulfonated polyanions: Nafion, poly(vinyl sulfonate) (PVS), and poly(styrene sulfonate) (PSS) serving as the counterion and providing an acidic microenvironment to stabilize PAni, are used to fabricate a sensor for ammonium ion detection. Nafion used to be a common ion-sensitive membrane due to its high proton conductivity. However, its high cost and limited solubility has constrained its uses. PVS and PSS are water-soluble polymers, easily incorporating with PAni to form the composites. Surface analysis by electron spectroscopy for chemical analysis (ESCA) and scanning electron microscope (SEM), and the electrochromic property for the PAni composites provided the convenient tools to characterize the electrode fabrication. On the aspect of sensing the ammonium ions, the modified electrodes exhibited electroactivity of PAni in ammonium ion detection and also showed the linear dependence of reduction current on the ammonium ion concentration. The pH effect on the sensing response was also evaluated and found insignificant to the response (ranging from pH 6.9-7.6). For increasing the stability of the electrodes, the diazo-resin (DAR) was introduced to the coat on the outmost layer and then cured by UV irradiation, giving the covalent network between the layers of polyelectrolytes. The PSS-doped PAni electrode was found to perform detection sensitivity in the linear range of 0-100mM of ammonium ion concentration.     

Coulombic and noncoulombic effect of polyanions on cytochrome c structure

The properties of the complexes of ferricytochrome c with two different polyanions—poly(vinylsulfate) and poly(4-styrene-sulfonate)—with a comparable charge density but with the different size of the uncharged part of their molecules have been studied by means of optical spectroscopy, differential scanning calorimetry, and gel chromatography. Ferriccytochrome c formed a complex with the former one through coulombic interactions and remained in a native-like state. The addition of the second polyanion to a solution of ferric cytochrome c at a low ionic strength, pH 7.0, resulted in profound conformational change in the hydrophobic core of protein (opening of the heme crevice with a perturbation of the methionine 80-heme iron bond and the hydrophobic core of the protein). These may be understood as an involvement of noncoulombic (hydrophobic, H-bonding) interactions of the uncharged part of the polyanion molecule. Conformational changes and the observed shift in acidic transition from low spin to high spin state of ferric cytochrome c detected in the presence of the polyanions may have biological implication in understanding the origin of conformational changes in proteins induced in the course of their interaction with membrane lipids and membrane proteins. © 1998 John Wiley & Sons, Inc. Biopoly 46: 145–154, 1998     

Protein-filled polyelectrolyte microcapsules in the design of enzymic microdiagnostics

Encapsulation of enzymes (lactate dehydrogenase and urease) in polyelectrolyte shells was assessed with a view to designing enzymic microdiagnostics for low-molecular compounds in native biological fluids. Polyelectrolyte microcapsules were prepared with two polyanions [poly(styrenesulfonate) PSS and dextran sulfate DS] and two polycations [poly(allylamine) PAA and poly(diallyldimethylammonium) PDADMA]; calcium carbonate microspherulites with embedded enzymes served as “cores.” It was demonstrated that the main problem in making such a biosensor is to select a pair of oppositely charged polyelectrolytes that would be optimal for enzyme functioning. The best pairs were PAA/DS and PAA/PSS for lactate dehydrogenase, and PSS/PAA and PSS/PDADMA for urease. We designed and prepared enzyme-containing microcapsules differing in polyelectrolyte composition and number of layers, and investigated their properties.     

Effect of poly(phosphate) anions on glyceraldehyde-3-phosphate dehydrogenase structure and thermal aggregation: comparison with influence of poly(sulfoanions)

Background

It is well documented that poly(sulfate) and poly(sulfonate) anions suppress protein thermal aggregation much more efficiently than poly(carboxylic) anions, but as a rule, they denature protein molecules. In this work, a polymer of different nature, i.e. poly(phosphate) anion (PP) was used to elucidate the influence of phosphate groups on stability and thermal aggregation of the model enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Methods

Isothermal titration calorimetry and differential scanning calorimetry were used for studying the protein–polyanion interactions and the influence of bound polyanions on the protein structure. The enzymatic activity of GAPDH and size of the complexes were measured. The aggregation level was determined from the turbidity.

Results

Highly polymerized PP chains were able to suppress the aggregation completely, but at significantly higher concentrations as compared with poly(styrenesulfonate) (PSS) or dextran sulfate chains of the same degree of polymerization. The effect of PP on the enzyme structure and activity was much gentler as opposed to the binding of dextran sulfate or, especially, PSS that denatured GAPDH molecules with the highest efficacy caused by short PSS chains. These findings agreed well with the enhanced affinity of polysulfoanions to GAPDH.

Conclusions

The revealed trends might help to illuminate the mechanism of control of proteins functionalities by insertion of charged groups of different nature through posttranslational modifications.

General significance

Practical implementation of the results could be the use of PP chains as promising tools to suppress the proteins aggregation without noticeable loss in the enzymatic activity.     

Effect of polyanion on the acidic conformational transition of native and denatured ferricytochrome c. Circular dichroism study

Interaction of polyanion poly(vinylsulfate) with oxidized cytochrome c (cyt c) significantly affects the protein main characteristics. One of them, pKa value of acidic transition, was shifted from an apparent pKa value 2.5 (typical for cyt c in low ionic strength solvent) to approximately 5.20 +/- 0.15 upon polyanion binding to the protein, pointing to a likely involvement of histidines 26 and/or 33 in the protein acidic transition in complex with the polyanion. The acidic transition followed at 6 different wavelengths all over circular dichroism spectrum, monitoring different parts of the protein structure, revealed basically two-state character process. Only ellipticity at 262 nm indicated a low-cooperative pH-induced conformational transition in heme region with an apparent pKa approximately 4.34 +/- 0.25 in accordance with absorbance change at 620 nm. Polyanion also interacts with chemically-denatured (in the presence of 9 mol/l urea) state of the protein as it follows from stabilization of protein residual structure at acidic pH and its effect on pKa value of acidic transition of chemically-denatured cyt c. Destabilization effect of polyanions on native and, on the other hand, stabilization influence on partially unfolded conformations of the protein are discussed with an implication for their chaperone-like properties in vivo and in vitro.     

The effect of complex-formation with polyanions on the redox properties of cytochrome c.

1. The stable complex formed between mammalian cytochrome c and phosvitin at low ionic strength was studied by partition in an aqueous two-phase system. Oxidized cytochrome c binds to phosvitin with a higher affinity than reduced cytochrome c. The difference was equivalent to a decrease of the redox potential by 22 mV on binding. 2. Complex-formation with phosvitin strongly inhibited the reaction of cytochrome c with reagents that react as negatively charged species, such as ascorbate, dithionite, ferricyanide and tetrachlorobenzoquinol. Reaction with uncharged reagents such as NNN'N'-tetramethylphenylenediamine and the reduced form of the N-methylphenazonium ion (present as the methylsulphate) was little affected by complex-formation, whereas oxidation of the reduced cytochrome by the positively charged tris-(phenanthroline)cobalt(III) ion was greatly stimulated. 3. A similar pattern of inhibition and stimulation of reaction rates was observed when phosvitin was replaced by other macromolecular polyanions such as dextran sulphate and heparin, indicating that the results were a general property of complex-formation with polyanions. A weaker but qualitatively similar effect was observed on addition of inositol hexaphosphate and ATP. 4. It is suggested that the effects of complex-formation with polyanions on the reactivity of cytochrome c with redox reagents are mainly the result of replacing the positive charge on the free cytochrome by a net negative charge. Any steric effects on polyanion binding are small in comparison with such electrostatic effects.     

Characterization of polyanion-protein complexes by frontal analysis continuous capillary electrophoresis and small angle neutron scattering: effect of polyanion flexibility

The binding constant (K(obs)) for the beta-lactoglobulin-poly(vinylsulfate) (BLG-PVS) complex was measured by frontal analysis continuous capillary electrophoresis at pH values above the isoelectric point of BLG, and the persistence length (L(p)) of PVS was measured by small angle neutron scattering, to examine the effect of polyelectrolyte chain stiffness on its binding efficiency to proteins. The values of K(obs) and L(p) were compared with those of BLG-PSS and BLG-PAMPS (poly(2-acrylamido-2-methylpropanesulfonate)) reported previously. The relationship between K(obs) and L(p) was reciprocal, indicating that protein binding is enhanced by the flexibility of the polyanion, at least in the case where the net protein charge is negative. In addition, at a fixed pH, the polymer systems displayed a similar ionic strength dependence of K(obs). This similarity was consistent with the proposal that the binding properties of PVS and PAMPS polyanions are governed purely by electrostatic interactions and are independent of their molecular structure.     

Calorimetric studies of the thermal denaturation of cytochrome c peroxidase

Two endotherms are observed by differential scanning calorimetry during the thermal denaturation of cytochrome c peroxidase at pH 7.0. The transition midpoint temperatures (tm) were 43.9 +/- 1.4 and 63.3 +/- 1.6 degrees C, independent of concentration. The two endotherms were observed at all pH values between 4 and 8, with the transition temperatures varying with pH. Precipitation was observed between pH 4 and 6, and only qualitative data are presented for this region. The thermal unfolding of cytochrome c peroxidase was sensitive to the presence and ligation state of the heme. Only a single endotherm was observed for the unfolding of the apoprotein, and this transition was similar to the high-temperature transition in the holoenzyme. Addition of KCN to the holoenzyme increases the midpoint of the high-temperature transition whereas the low-temperature transition was increased upon addition of KF. Binding of the natural substrate ferricytochrome c to the enzyme increases the low-temperature transition by 4.8 +/- 1.3 degrees C but has no effect on the high-temperature transition at pH 7. The presence of cytochrome c peroxidase decreases the stability of cytochrome c, and both proteins appear to unfold simultaneously. The results are discussed in terms of the two domains evident in the X-ray crystallographic structure of cytochrome c peroxidase.     

Conformation of poly(L-arginine). II. Complexes with polyanions

Conformaitons of poly(L -arginine)/polyanion complexes were studies by CD measurements. The polyanions were the homoplolypeptides poly(L -glutamic acid) and poly(L -aspartic acid); the synthetic polyelectrolytes and polyethylenesulfonate; and the polynucleotides were native DNA, denatured DNA, and poly(U). It was found that poly(L -arginine) forms the α-helical conformation by interacting with the acidic homopolypeptides and the synthetic anionic polyelectrolytes. In each complex, poly(L -glutamic acid) is in the α-helical conformation, whereas poly(L -aspartic acid) is mostly in the random structure. The poly(L -glutamic acid) complex changed into the β-sheet structure at the transition temperature about 65°C in 0.01M cacodylate buffer (pH 7). Even in the presence of 5M urea, this complex remained in the α-helical conformation at room temperature. The existence of the stable complex of α-helical poly(L -arginine) and α-helical poly(L -glutamic acid) was successfully supported by the model building study of the complex. The α-helix of poly(L -arginine) induced by binding with polyacrylate was the most stable of the poly(L -arginine)-polyanion complexes examined as evidenced by thermal and urea effects. The lower helical content of the polyethylenesulfonate-complexed poly(L -aginine) was explained in terms of the higher charge density of the polyanion. On the other hand, native DNA, denatured DNA, and poly(U) were not effective in stabilizing the helical structure of poly(L -arginine). This may be due to the rigidity of polyanions and to the steric hindrance of bases. Furthermore, the distinitive structual behavior of poly(L -arginine) and poly(L -lysine) regarding polyanion interaction has been noticed throughout the study.     

Protein and lipid structural transitions in cytochrome c oxidase-dimyristoylphosphatidylcholine reconstitutions

The thermotropic behavior of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) reconstituted in dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by using high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome c oxidase into the phospholipid bilayer perturbs the thermodynamic parameters associated with the lipid phase transition in a manner analogous to other integral membrane proteins: it reduces the enthalpy change, lowers the transition temperature, and reduces the cooperative behavior of the phospholipid molecules. Analysis of the dependence of the enthalpy change on the protein:lipid molar ratio indicates that cytochrome c oxidase prevents 99 +/- 5 lipid molecules from participating in the main gel-liquid-crystalline transition. These phospholipid molecules presumably remain in the same physical state below and above the transition temperature of the bulk lipid, thus providing a more or less constant microenvironment to the protein molecule. The effect of the phospholipid bilayer matrix on the thermodynamic stability of the cytochrome c oxidase complex was examined by high-sensitivity differential scanning calorimetry. Detergent (Tween 80)-solubilized cytochrome c oxidase undergoes a complex, irreversible thermal denaturation process centered at 56 degrees C and characterized by an enthalpy change of 550 +/- 50 kcal/mol of enzyme complex. Reconstitution of the cytochrome c oxidase complex into DMPC vesicles shifts the transition temperature upward to 63 degrees C, indicating that the phospholipid bilayer moiety stabilizes the native conformation of the enzyme. The lipid bilayer environment contributes approximately 10 kcal/mol to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome c oxidase is not a two-state process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthetic and natural polyanions induce cytochrome c release from mitochondria in vitro and in situ

A synthetic polyanion composed of styrene, maleic anhydride, and methacrylic acid (molar ratio 56:37:7) significantly inhibited the respiration of isolated rat liver mitochondria in a time-dependent fashion that correlated with 1) collapse of the mitochondrial membrane potential and 2) high amplitude mitochondrial swelling. The process is apparently Ca(2+) dependent. Since it is blocked by cyclosporin A, the process is ascribed to induction of the mitochondrial permeability transition. In mitoplasts, i.e., mitochondria lacking their outer membranes, the polyanion rapidly blocked respiration. After incubation of rat liver mitochondria with the polyanion, cytochrome c was released into the incubation medium. In solution, the polyanion modified by conjugation with fluorescein formed a complex with cytochrome c. Addition of the polyanion to cytochrome c-loaded phosphatidylcholine/cardiolipin liposomes induced the release of the protein from liposomal membrane evidently due to coordinated interplay of Coulomb and hydrophobic interactions of the polymer with cytochrome c. We conclude that binding of the polyanion to cytochrome c renders it inactive in the respiratory chain due to exclusion from its native binding sites. Apparently, the polyanion interacts with cytochrome c in mitochondria and releases it to the medium through breakage of the outer membrane as a result of severe swelling. Similar properties were demonstrated for the natural polyanion, tobacco mosaic virus RNA. An electron microscopy study confirmed that both polyanions caused mitochondrial swelling. Exposure of cerebellar astroglial cells in culture to the synthetic polyanion resulted in cell death, which was associated with nuclear fragmentation.     

Differential susceptibilities of DNA polymerases-alpha and -beta to polyanions.

The effects of various polyanions including synthetic polynucleotides on DNApolymerases-alpha and -beta from blastulae of the sea urchin Hemicentrotus pulcherrimus and HeLa cells were studied. Only DNA polymerase-alpha was inhibited by polyanions, such as polyvinyl sufate, dextran sulfate, heparin, poly(G), poly(I), poly(U) and poly(ADP-Rib). Of the various polynucleotides tested, poly(G) and poly(I) were the strongest inhibitors. Kinetic studies showed that the Ki value for poly(G) was 0.3 microgram/ml and that poly(G) had 20-fold higher affinity than activated DNA for the template-primer site of DNA polymerase-alpha. Poly(U) and poly(ADP-Rib) were also inhibitory, but they were one hundredth as inhibitory as poly(G) or poly(I). Poly(A), poly(C), poly(A).poly(U) AND POLY(I).poly(C) were not inhibitory to DNA polymerase-alpha. In contrast, DNA olymerase-beta was not affected at all by these polyanions under the same conditions.     

Modification of the structural and redox properties of cytochrome c by heteropolytungstate binding

Complex formation between horse heart ferricytochrome c and large three-dimensional polyanions has been investigated, in order to study the influence of surface electrostatic interactions on the structural and redox properties of cytochrome c. Cytochrome c binds the large heteropolytungstates (NaSb9W21O86)18- and (KAs4W40O140)27- with a 1/1 polyanion/cytochrome c ratio, and the smaller ion (SiW11O39)8- with a 2/1 ratio. Upon complexation, cytochrome c undergoes structural changes that are dependent on the size and charge of the polyanion, and on the pH and ionic strength of the medium. Three different forms of complexed cytochrome c have been characterized by optical and EPR spectroscopies, in the pH range 6.5-8: an N form, close to the native structure, an A form, analogous to cytochrome c in acidic medium, and a novel B form in which the heme pocket is open but the iron remains low-spin. The redox potential of cytochrome c is lowered to 250-220 mV (vs. NHE) in the N form, and to 80 mV in the B form.     

Enzyme behaviour and molecular environment. The effects of ionic strength, detergents, linear polyanions and phospholipids on the pH profile of soluble cytochrome oxidase.

The activity vs. pH profile for the oxidation of ferrocytochrome c by purified cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was investigated as a function of ionic strength (from 10 to 200 mM) in the absence and in the presence of various perturbants: Tween 20, linear polyanions (RNA, heparin, polyglutamic acid) and phospholipids (asolectin, phosphatidylcholine, phosphatidic acid and cardiolipin). The activation induced by Tween 20 and "zero net charge" phospholipid liposomes was not pH dependent. On the other hand, linear polyanions and polyanionic liposomes strongly perturbed the pH profile, mostly at low ionic strength, by shifting the pH optimum about 1.7 pH units towards alkaline pH values. This effect was reversed by increasing ionic strength. These observations are interpreted in the light of polyelectrolyte theory. Since these results show striking with membrane-bound enzyme, it is concluded that in vivo cytochrome oxidase is located within polyanionic sites of the micochondrial membrane. The activation broght about by phospholipids may result from two posible processes: creation of a hydrophobic environment by the non-polar tails, preventing autoaggregation; and creation of a suitable polyelectrolytic environment by the polar heads (of non zero net charge), increasing the intrinsic reaction rate.     

Inhibition of pepsin by polyions and c.d. studies

The effects of poly(vinyl sulphate) (PVS), poly(vinyl pyrrolidone) (PVP) and protamine sulphate with the enzyme pepsin (EC 3.4.23.1) have been investigated. PVS, PVP and protamine acted as inhibitors of the enzyme pepsin at low concentrations, but at high concentrations of the polyions (with the exception of PVS) the inhibition was less pronounced. The catalytic effectiveness of several polyions has been shown experimentally; when used at pH 2.1 with haemoglobin and at pH 5.0 with azocasein thus acted as a weak proteolytic catalyst. The order of effectiveness was: polybrene greater than poly(L-lysine) (PLL) greater than spermine greater than spermidine greater than protamine greater than and PVP relative to pepsin. The hydrolysis of the substrates by the enzyme decreases in the presence of high concentrations of monovalent and/or divalent salts. The purity of the enzyme was assessed by determination of the molecular weight using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), phosphorus and nitrogen content. The circular dichroism (c.d.) spectrum of the enzyme in the wavelength range 240-310 nm, where the polycations did not have a c.d. spectrum has also been studied. Each of the polyions had a definite effect on the c.d. spectrum, showing strong binding to the enzyme. The neutral polymer PVP was also found to modify the c.d. spectrum, showing strong binding to the enzyme. The strengths of the interactions as indicated by the magnitudes of the changes in the c.d. spectrum of pepsin at 270 nm were: polybrene greater than protamine greater than PLL greater than PVP greater than spermidine greater than spermine.     

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 citrate-ferrocytochrome 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 Km (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 Km of 170 microM at very high ionic strength indicates a ratio of K infinity m/K 0 M of about 5000. It is proposed that anions either modify the E10 of cytochrome C bound at the primary (T) site of that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

Core--shell nanocluster films of hemoglobin and clay nanoparticle: direct electrochemistry and electrocatalysis

A novel core-shell protein nanocluster film, designated as clay-(Hb/PSS)(n), was fabricated on pyrolytic graphite (PG) electrodes. Positively charged hemoglobin (Hb) at pH 5.5 and negatively charged poly(styrenesulfonate) (PSS) were first assembled layer by layer on surface of clay nanoparticles from their solutions mainly by electrostatic attraction, forming a core-shell nanocluster structure in which clay nanoparticles were the "cores" and (Hb/PSS)(n) multilayers were the "shells". The aqueous dispersion of clay-(Hb/PSS)(n) nanoclusters was then cast on surface of PG electrodes, forming clay-(Hb/PSS)(n) nanocluster films after evaporation of solvent. Hb in clay-(Hb/PSS)(n) films exhibited a pair of well-defined and reversible cyclic voltammetric (CV) peaks at about -0.36 V vs. SCE in pH 7.0 buffers, characteristic of Hb heme Fe(III)/Fe(II) redox couples. Compared with other Hb-containing clay films, clay-(Hb/PSS)(n) films displayed smaller CV peak separation (DeltaE(p)), indicating the better electrochemical reversibility of Hb in these nanocluster films. The partially ordered structure of the films was characterized by X-ray diffraction (XRD) experiments. UV-VIS and reflection absorption infrared (RAIR) spectroscopy suggests that Hb retains its near-native structure in clay-(Hb/PSS)(n) films. Oxygen, hydrogen peroxide, and nitrite were catalytically reduced at clay-(Hb/PSS)(n) film electrodes, showing the potential applicability of the films as the new type of biosensors or bioreactors based on protein direct electrochemistry. The electrochemical and electrocatalytic activity of the films could be tailored by controlling the number of bilayers of the (Hb/PSS)(n) shells on the surface of clay nanoparticle cores.     

Charge-dependent sidedness of cytochrome P450 forms studied by quartz crystal microbalance and atomic force microscopy

Quartz crystal microbalance (QCM) resonance measurements were used to examine the surface charge characteristics of cytochrome P450 forms and the influence of charge on the docking of redox partners like cytochrome b5. The distal surface of cytochrome P450 (CYP)101 (pI = 4.5), relative to the heme, is fairly anionic, as is the proximal surface. The latter, however, also has two cationic clusters. A considerably greater extent of CYP101 binding was seen to the cationic, polyethylene-surfaced resonators. CYP2B4 (pI = 8.5) preferentially bound to the polyanionic, polystyrene sulfonate-surfaced resonators. Cytochrome b5 is an acidic protein that had a preferential binding to the poly(ethyleneimine (PEI)-surfaced resonators. When binding to CYP2B4-surfaced films, cytochrome b5 preferentially bound to those cytochrome P450 molecules that were adsorbed to cationic (PEI) films. It is suggested that adsorption of CYP2B4 to an anionic poly(styrenesulfonate) (PSS) surface is with cationic clusters that include the cytochrome b5 docking domain. This diminishes the extent of docking of the cytochrome b5. In contrast, when CYP2B4 is adsorbed to a cationic film the proximal surface with the cytochrome b5-docking site is available for cytochrome b5 binding. A film of the polycation PEI was adsorbed to the silver QCM surface. It formed polymer islands when viewed with atomic force microscopy. Polyanionic PSS was adsorbed intermittently with the PEI. By the third and fourth layer of polyions the polymer islands were essentially merged and protein adsorption as a fourth or fifth layer formed a nearly continuous film. CYP101 was seen to adsorb as globules with a molecular diameter of about 10 nm. CYP2B4 adsorbed to the polyionic films had a slightly elliptical globular shape, also with a molecular diameter of about 10 nm.