Changes In Surface Hydrophobicity Research Articles

Cytochrome P450 52A3: Modelling of 3D Structure and Surface Mutations

Earlier W.-H. Schunck et al. [1] have prepared a water soluble enzymatically active fragment of cytochrome P450 52A3 (CYP52A3) which is lack of 66 amino acid residues, existed as a dimer in aqueous solution. Now we propose 3D structure of the fragment, which is based on multiple sequence alignment of the CYP52A3 with its homologues proteins of known 3D structure: CYP101, 102, 107A1 and 108. The structural model have been optimised and used as a prototype for computer simulation of point mutations. These mutations should bring some changes in the surface properties, interfering dimer formation. For this aim the point of 22 hydrophobic amino acid residues have been sequentially replaced with that of charged amino acids (GLU, ASP, ARG and LYS). The scoring of “mutants” was conducted based on the changes of protein surface hydrophobicity and protein-solvent interaction energy. An analysis of the surface hydrophobicity and protein-solvent interactions permit to select most sensitive three sites (171, 352 and particularly 164 amino acid residues). The dimerization of the following “mutant” fragments must be investigated experimentally.

Direct Characterization of the Physicochemical Properties of Fungal Spores Using Functionalized AFM Probes

A new method is described for characterizing the physicochemical properties of native microbial cells by using atomic force microscopy (AFM) with chemically functionalized probes. Adhesion forces were measured, under deionized water, between probes and model substrata functionalized with alkanethiol self-assembled monolayers terminated with OH and CH 3 groups. These were found to be 6 ± 2 nN ( n = 1024), 0.9 ± 0.4 nN, and ∼0 nN, for CH 3/CH 3, CH 3/OH, and OH/OH surfaces, respectively, and were not significantly influenced by changes of ionic strength (0.1 M NaCl versus deionized water). This shows that functionalized probes are very sensitive to changes of surface hydrophobicity. Using OH- and CH 3-terminated probes, patterns of rodlets, ∼10 nm in diameter, were visualized, under physiological conditions, at the surface of spores of Phanerochaete chrysosporium. Multiple (1024) force-distance curves recorded over 500 × 500-nm areas at the spore surface, either in deionized water or in 0.1 M NaCl solutions, always showed no adhesion for both OH- and CH 3-terminated probes. Control experiments indicated that the lack of adhesion is not due to transfer of cellular material onto the probe, but to the hydrophilic nature of the spore surface.

Temperature-dependent Chaperone Activity and Structural Properties of Human αA- and αB-crystallins

The chaperone activity and biophysical properties of recombinant human alphaA- and alphaB-crystallins were studied by light scattering and spectroscopic methods. While the chaperone function of alphaA-crystallin markedly improves with an increase in temperature, the activity of alphaB homopolymer appears to change very little upon heating. Compared with alphaB-crystallin, the alphaA-homopolymer is markedly less active at low temperatures, but becomes a more active species at high temperatures. At physiologically relevant temperatures, the alphaB homopolymer appears to be modestly (two times or less) more potent chaperone than alphaA homopolymer. In contrast to very similar thermotropic changes in the secondary structure of both homopolymers, alphaA- and alphaB-crystallins markedly differ with respect to the temperature-dependent surface hydrophobicity profiles. Upon heating, alphaA-crystallin undergoes a conformational transition resulting in the exposure of additional hydrophobic sites, whereas no such transition occurs for alphaB-crystallin. The correlation between temperature-dependent changes in the chaperone activity and hydrophobicity properties of the individual homopolymers supports the view that the chaperone activity of alpha-crystallin is dependent on the presence of surface-exposed hydrophobic patches. However, the present data also show that the surface hydrophobicity is not the sole determinant of the chaperone function of alpha-crystallin.

Mucin Coating on Hydrophobic Polymer Materials

AbstractIn this work, the adsorption isotherm and kinetics of bovine submaxillary gland mucin (BSM) onto a hydrophobic polystyrene surface were studied by the solution depletion method, in which mucin surface concentrations were analyzed by amino acid analysis. Using a Langmuir adsorption model and non-linear curve fitting, kinetics parameters, kon and koff were determined. The coating was found to be very stable with very limited desorption (less than 2%) from a long term observation. By measuring the water contact angles, the changes in surface hydrophobicity due to mucin coating were monitored on four polymer materials, namely polymethylmethacrylate (PMMA), polyurethane (PU), polystyrene (PS), and silicone. After coating, all the hydrophobic surfaces turned into very hydrophilic. A strict correlation between mucin surface concentration and surface wettability has been found.

Attenuation of hydrophobic phospholipid barrier is an early event in Helicobacter felis-induced gastritis in mice.

Helicobacter pylori infection has been linked to the development of gastritis which can then progress to a number of disease entities including peptic ulcer disease and gastric cancer. Since the pathogenic mechanism by which the bacteria causes gastritis is unresolved, we employed a model system, the H. felis-infected mouse to investigate the temporal relationship between bacterially-induced alterations in the hydrophobic phospholipid barrier of the stomach and the development of gastritis. In the present study, C57BL/6 mice were inoculated with 10(9) CFU of H. felis and the changes in gastric wet weight, histology, surface hydrophobicity, phospholipid/phosphatidylcholine concentration, phospholipase A2 activity, and the pH of collected gastric juice were measured 0.5-2 months postinoculation. In related experiments, we investigated the effects of treating H. felis infected mice with antibiotic/ bismuth therapy on the above gastric properties. It was determined that both gastric surface hydrophobicity and phospholipid composition were significantly attenuated as early as 2-4 weeks postinfection, preceding signs of mucosal inflammation and glandular atrophy as indicated by increases in gastric wet weight, pH and a disappearance in parietal cells. These early H. felis-induced changes in gastric surface hydrophobicity and phospholipid concentration were reversed by antibiotic/bismuth therapy. Based on these results we conclude that H. felis infection induces an early transformation of the stomach from a hydrophobic to an acid-sensitive hydrophilic state that may trigger the subsequent development of gastritis.

Coenzyme Q10 in vesicles composed of archaeal ether lipids or conventional lipids enhances the immuno-adjuvanticity to encapsulated protein

Cellular accumulation, tissue distribution, and immuno-adjuvanticity were evaluated for liposomal CoQ10 prepared from either distearoylphosphatidylcholine:dicetylphosphate:cholesterol (4:1:5, mol. ratio) (conventional liposomes) or from the total polar lipids of the archaeon Methanosarcina mazei (archaeosomes). Liposomal CoQ10 vesicles of approximately 100 nm diameter, containing up to 179 μmol of CoQ10 per mg of lipid have been evaluated using J774A.1 macrophages and Balb/c mice. Archaeosomes uptake by J774A.1 macrophages was better than with the conventional liposome, and the incorporation of CoQ10 enhanced the uptake of both lipid vesicle types. All vesicle types were detected in the liver and spleen of mice (4–27% of injected dose) within 3 h of intraperitoneal injection. Moreover, incorporation of CoQ10 into lipid vesicles enhanced the immuno-adjuvanticity of both conventional liposomes and archaeosomes, to achieve approximately a doubling in the titres of BSA-specific antibody in sera to 169 and 430 μg ml −1, respectively. Increases in IgG1 and IgG2a/2b accounted for most of the CoQ10-induced increases in anti-BSA titres. These results are rationalized on the basis of surface hydrophobicity and opsonization changes induced by the presence of CoQ10 in vesicles. We suggest that liposomal CoQ10 has potential as a new generation of vaccine delivery system to enhance the immune response. Its use as a novel delivery system may be particularly effective under pathological conditions where the occurrence of an oxidative stress condition significantly impairs the immune system functions.

Refolding of urea-denatured adenylate kinase.

The refolding of urea-denatured adenylate kinase (EC 2.7.4.3) has been followed by formation of the secondary structure, change of surface hydrophobicity and recovery of catalytic activity. During refolding of adenylate kinase with a 20-80-fold dilution of 4 M urea-denatured enzyme at 10 degrees C, the formation of the secondary structure is a fast process with a rate constant of >0.16 s-1. Transient enhancement of the 8-anilino-1-naphthalenesulphonate (ANS) fluorescence intensity is followed by a fluorescence decrease to the level equal to the value characteristic of native enzyme. The desorption of ANS binding fluorescence is relatively slow and can be fitted to a first order reaction with a rate constant of 0.004 s-1 when the ANS is present in the dilution buffer. The desorption of ANS-binding fluorescence is accelerated in the presence of nucleotide substrates. The rate constants are increased to 0.049, 0. 029, 0.028 and 0.029 s-1 in the presence of 1 mM AMP, MgATP, ATP and ADP respectively. The refolding rate constant calculated from the initial fluorescence intensity after mixing ANS with protein at different refolding intervals is 0.016 s-1, which is faster than those obtained when ANS is present throughout the refolding process, indicating that the binding of ANS with a partially folded intermediate retards its further refolding to its native structure. The reactivation rate is even faster than the rates of refolding monitored in the absence of substrates, showing that the refolding is accelerated in the presence of the substrates. A possible refolding pathway and the accelerating effect of substrates are discussed.

Biodegradation of blends of bacterial poly(3-hydroxybutyrate) with ethyl cellulose in activated sludge and in enzymatic solution

Blends of poly(3-hydroxybutyrate) (PHB) and ethyl cellulose (EtC) were prepared by compression molding of solution cast films. The two components were not miscible but the blends showed ‘mechanical compatibility’. Two PHB/EtC blends (80/20 and 50/50 (w/w in all cases)) were selected for biodegradation experiments in activated sludge and in enzymatic solution of PHB-depolymerase from Pseudomonas lemoignei and from Aureobacterium saperdae. Blend morphology was quite different: blend 80/20 was composed of a matrix of impinging PHB spherulites with dispersed EtC inclusions, whereas blend 50/50 was constituted of two continuous phases with interpenetrated domains. Both blends biodegraded in activated sludge, but only blend 80/20 was attacked by PHB-depolymerases from P. lemoignei and A. saperdae. From weight loss, thickness and composition measurements after different exposure times and from scanning electron microscopy of the biodegraded blend surface it was concluded that in blend 80/20 biodegradation of the PHB matrix caused concomitant release in the medium of the embedded EtC particles. The results on PHB/EtC (50/50) after sludge exposure demonstrated that in this blend the PHB phase – thanks to its interconnected domains – was continuously accessible to microbial attack, leaving a highly cavitated ethyl cellulose structure after PHB consumption. The lack of biodegradation of blend 50/50 in enzymatic solution was tentatively attributed to changes of surface hydrophobicity induced by large amounts of EtC, which disfavored enzyme binding.

Biodegradation of blends of bacterial poly(3-hydroxybutyrate) with ethyl cellulose in activated sludge and in enzymatic solution

Blends of poly(3-hydroxybutyrate) (PHB) and ethyl cellulose (EtC) were prepared by compression molding of solution cast films. The two components were not miscible but the blends showed ‘mechanical compatibility’. Two PHB/EtC blends (80/20 and 50/50 (w/w in all cases)) were selected for biodegradation experiments in activated sludge and in enzymatic solution of PHB-depolymerase from Pseudomonas lemoignei and from Aureobacterium saperdae. Blend morphology was quite different: blend 80/20 was composed of a matrix of impinging PHB spherulites with dispersed EtC inclusions, whereas blend 50/50 was constituted of two continuous phases with interpenetrated domains. Both blends biodegraded in activated sludge, but only blend 80/20 was attacked by PHB-depolymerases from P. lemoignei and A. saperdae. From weight loss, thickness and composition measurements after different exposure times and from scanning electron microscopy of the biodegraded blend surface it was concluded that in blend 80/20 biodegradation of the PHB matrix caused concomitant release in the medium of the embedded EtC particles. The results on PHB/EtC (50/50) after sludge exposure demonstrated that in this blend the PHB phase – thanks to its interconnected domains – was continuously accessible to microbial attack, leaving a highly cavitated ethyl cellulose structure after PHB consumption. The lack of biodegradation of blend 50/50 in enzymatic solution was tentatively attributed to changes of surface hydrophobicity induced by large amounts of EtC, which disfavored enzyme binding.

Biodegradation of blends of bacterial poly(3-hydroxybutyrate) with ethyl cellulose in activated sludge and in enzymatic solution

Blends of poly(3-hydroxybutyrate) (PHB) and ethyl cellulose (EtC) were prepared by compression molding of solution cast films. The two components were not miscible but the blends showed ‘mechanical compatibility’. Two PHB/EtC blends (80/20 and 50/50 (w/w in all cases)) were selected for biodegradation experiments in activated sludge and in enzymatic solution of PHB-depolymerase from Pseudomonas lemoignei and from Aureobacterium saperdae. Blend morphology was quite different: blend 80/20 was composed of a matrix of impinging PHB spherulites with dispersed EtC inclusions, whereas blend 50/50 was constituted of two continuous phases with interpenetrated domains. Both blends biodegraded in activated sludge, but only blend 80/20 was attacked by PHB-depolymerases from P. lemoignei and A. saperdae. From weight loss, thickness and composition measurements after different exposure times and from scanning electron microscopy of the biodegraded blend surface it was concluded that in blend 80/20 biodegradation of the PHB matrix caused concomitant release in the medium of the embedded EtC particles. The results on PHB/EtC (50/50) after sludge exposure demonstrated that in this blend the PHB phase – thanks to its interconnected domains – was continuously accessible to microbial attack, leaving a highly cavitated ethyl cellulose structure after PHB consumption. The lack of biodegradation of blend 50/50 in enzymatic solution was tentatively attributed to changes of surface hydrophobicity induced by large amounts of EtC, which disfavored enzyme binding.

Effect of maturity and curing on peanut proteins. Changes in protein surface hydrophobicity.

A hydrophobic fluorescence probe, 1,8-anilinonaphthalene sulfonate (ANS), was used to study the changes in protein surface hydrophobicity (PSH) occurring during peanut maturation and curing. PSH increased with the degree of maturity and during curing (windrow drying). The increase of PSH during curing or heating was more pronounced in immature peanuts than their mature counterparts, suggesting that more hydrophobic sites are hidden in the former proteins. PSH decreased when proteins were chemically modified with phenylglyoxal (an arginine-modifying agent), suggesting that arginine might play a role in hydrophobicity. The findings indicate that maturation and curing affect PSH, and that there is a relationship between PSH and peanut maturity. Possible factors contributing to the increase of PSH are discussed.

Effects of flour chlorination on soft wheat gliadins analyzed by reversed-phase high-performance liquid chromatography, differential scanning calorimetry and fluorescence spectroscopy

Chlorinated soft wheat flours produce better quality cakes. In this study, changes in surface hydrophobicity of gliadins due to flour chlorination were analyzed. Results obtained from RP-HPLC of gliadins showed intervarietal differences, but intravarietal differences in the gliadin patterns of chlorinated and unchlorinated flours were difficult to discern because of the large number of peaks eluted. However, three-dimensional plots of retention time, wavelength and absorbency showed differences among gliadin patterns of unchlorinated and chlorinated flours within a variety, suggesting conformational changes and consequently alteration in hydrophobicity of gliadins due to chlorination. The DSC analyses of gliadins extracted from unchlorinated and chlorinated flours showed higher denaturation peak transition temperatures ( T d) and lower enthalpies of transition (Δ H) in some varieties, indicating hydrophobicity and conformational changes. Fluorescence spectroscopy measurements with an extrinsic probe, 1-anilino-8-naphthalene sulfonic acid (ANS), showed increases in hydrophobicity of gliadins from chlorinated flours, confirming conformational changes in gliadins due to chlorination.

Modification of protein surface hydrophobicity and methionine oxidation by oxidative systems.

Aging and some pathological conditions are associated with the accumulation of altered (inactive or less active) forms of enzymes. It was suggested that these age-related alterations reflect spontaneous changes in protein conformation and/or posttranslational modifications (e.g., oxidation). Because changes in protein conformations are often associated with changes in surface hydrophobicity, we have examined the effects of aging and oxygen radical-dependent oxidation on the hydrophobicity of rat liver proteins. As a measure of hydrophobicity, the increase in fluorescence associated with the binding of 8-anilino-1-naphthalene-sulfonic acid to hydrophobic regions on the proteins was used. By this criterion, the hydrophobicity of liver proteins of 24-month-old rats was 15% greater than that of 2-month-old animals. Exposure of liver proteins to a metal-catalyzed oxidation system (ascorbate/Fe(II)/H2O2) or a peroxyl radical generating system, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) led to increases of 2% or 30% in surface hydrophobicity, respectively. Treatment of liver proteins with the metal-catalyzed oxidation system led to a significant increase in reactive carbonyl content and to conversion of methionine residues to methionine sulfoxide residues. Treatment with AAPH led also to oxidation of methionine, tyrosine, and tryptophan residues and to the precipitation of some proteins. Dityrosine was detected in AAPH-treated protein, both the precipitate and supernatant fraction. The oxidation-dependent increase of hydrophobicity was correlated with an increase in the levels of methionine sulfoxide and dityrosine. These results suggest that oxidative modification of proteins may be responsible for the age-related increase of protein surface hydrophobicity in vivo, and that the oxidation of methionine by an oxidative system may be an important event for the change of protein conformation.

CHANGES IN STRATUM CORNEUM-WATER INTERACTIONS FOLLOWING BIRTH IN THE NEWBORN INFANT • 1021

The outermost layer of the skin, the stratum corneum (SC), is a thin, flexible, polymeric envelope required for survival in a terrestrial environment. Evidence from newborn animals demonstrates that the SC manifests a range of unique water interactions at birth including a hydrophobic surface mantle, integral barrier lipid lamellae, and spontaneous proteolysis of structural proteins, such as filaggrin, to form smaller water binding molecules necessary for polymer plasticization. In the present study, we evaluated the background of these interactions by quantifying changes in SC hydration, water flux, and surface hydrophobicity (water desorption) over the first 16h of life in 86 term infants. Unit measurement of surface electrical capacitive reactance (cru) served as the primary index of hydration state(Pediatrics 96:688-692, 1995). A separate study looking at the effect of occlusion was also performed using a plastic thermal wrap to occlude the lower body after birth. The results demonstrate 1) a progressive fall in surface hydration from 2h to 16 h after birth (113±3 to 105±5 cru's, respectively, p < 0.05), 2) a decrease in transepidermal water movement measured as the slope of the continuous capacitance curves(0.96±0.12 at 2h of age to 0.32±0.09 cru's/sec at 16h, p < 0.01), 3) an increase in surface water desorption (-7±0.6 at 2h of age to -12±1 cru's/sec × 1000 at 16h, p < 0.01); 4) higher baseline hydration following occlusion of the suprapubic region at 2h of age compared to the chest (135±7 to 120±4 cru's, respectively, p<0.01); and 5) increased transepidermal water movement following occlusion(2.86±0.34 compared to 1.21±0.16 cru's/sec, p<0.01).

Coupling of protein surface hydrophobicity change to ATP hydrolysis by myosin motor domain

Dielectric spectroscopy with microwaves in the frequency range between 0.2 and 20 GHz was used to study the hydration of myosin subfragment 1 (S1). The data were analyzed by a method recently devised, which can resolve the total amount of water restrained by proteins into two components, one with a rotational relaxation frequency (fc) in the gigahertz region (weakly restrained water) and the other with lower fc (strongly restrained water). The weight ratio of total restrained water to S1 protein thus obtained (0.35), equivalent to 2100 water molecules per S1 molecule, is not much different from the values (0.3-0.4) for other proteins. The weakly restrained component accounts for about two-thirds of the total restrained water, which is in accord with the number of water molecules estimated from the solvent-accessible surface area of alkyl groups on the surface of the atomic model of S1. The number of strongly restrained water molecules coincides with the number of solvent-accessible charged or polar atoms. The dynamic behavior of the S1-restrained water during the ATP hydrolysis was also examined in a time-resolved mode. The result indicates that when S1 changes from the S1.ADP state into the S1.ADP.P1 state (ADP release followed by ATP binding and cleavage), about 9% of the weakly restrained waters are released, which are restrained again on slow P1 release. By contrast, there is no net mobilization of strongly restrained component. The observed changes in S1 hydration are quantitatively consistent with the accompanying large entropy and heat capacity changes estimated by calorimetry (Kodama, 1985), indicating that the protein surface hydrophobicity change plays a crucial role in the enthalpy-entropy compensation effects observed in the steps of S1 ATP hydrolysis.

Surface composition, surface properties, and adhesiveness ofAzospirillum brasilense—variation during growth

The surface chemical composition, the physicochemical properties, and the adhesiveness of Azospirillum brasilense have been investigated during growth in Luria–Bertani* rich medium. The surface elemental composition obtained by X-ray photoelectron spectroscopy was converted into a molecular composition in terms of model constituents: proteins, polysaccharides, and hydrocarbon-like compounds. The protein content increased during growth, from 30 (exponential phase cells) to 50% (stationary phase cells), concomitantly with a decrease in the polysaccharide content, from 60 to 35%. These modifications were related to a change in cell surface hydrophobicity, i.e., to an increase of the water contact angle from 20 to 60°. No difference of electrophoretic mobility was detected between cells harvested in the exponential phase and cells harvested in the stationary phase. The increase of both cell surface protein concentration and cell surface hydrophobicity during growth was correlated with an increase of cell adhesiveness to model supports. This points to the involvement of cell surface proteins and cell surface hydrophobicity in the adhesion process.Key words: Azospirillum brasilense, surface composition, hydrophobicity, adhesiveness, X-ray photoelectron spectroscopy.

P-F1-22 Chemomechanical implication of surface hydrophobicity change in myosin motor domain (S1) revealed by dielectric spectroscopy: KODAMA T, 1 SUZUKI M 2 Y 1Kyushu Inst. Technology, Ihzuku, (Japan), 2NAIR/MEL, AIST, Tsukuba (Japan)

P-F1-22 Chemomechanical implication of surface hydrophobicity change in myosin motor domain (S1) revealed by dielectric spectroscopy: KODAMA T, 1 SUZUKI M 2 Y 1Kyushu Inst. Technology, Ihzuku, (Japan), 2NAIR/MEL, AIST, Tsukuba (Japan)

Changes in structural and antigenic properties of proteins by radiation

Radiation effect on structural and antigenic properties of proteins (0.2% in 0.01 M phosphate buffer, pH 7.4) were investigated using ovalbumin (OVA) and bovine serum albumin (BSA). Aggregation of OVA and BSA was induced by radiation and the molecular mass increased significantly in N 2. Significant changes in surface hydrophobicity and [ θ] 222 nm of CD were also observed by radiation showing the destruction of secondary structure of proteins. Antigenicity of irradiated OVA measured by the method of immunodiffusion was decreased by radiation, and the reactivity to anti-OVA antibody was almost diminished at 8 kGy in N 2 and 4 kGy in O 2, respectively. The reactivity of BSA was diminished at 4 kGy both in N 2 and O 2. Changes in hydrophobicity of OVA did not correspond to the decrease in antigenicity, whereas the changes in [ θ] 222 nm relatively well corresponded to the antigenicity. The SDS-PAGE and immunoblotting analysis showed that radiation at higher doses induced the production of protein aggregates and degraded fragments with reactivity to the specific antibodies. These results suggest that the main part of conformation-dependent antigenic structure (conformational epitope) is easily lost by radiation, but some antigenicity, which is mostly due to the amino acid sequence-dependent antigenic structures (sequential epitopes), remains even at higher dose.

Comments on fluorescence methods for probing local deviations from lamellar packing

Fluorescence probes have been used to monitor the conversion from the lamellar to nonlamellar phases. In addition there is current interest in understanding the nature of the changes in the physical properties of phospholipid bilayers that are correlated with their propensity for forming nonlamellar phases. Fluorescent probes have been used to demonstrate changes in surface hydrophobicity, quenching efficiency, and rates of interbilayer transfer of fluorescently labeled analogs. These studies are beginning to provide an assessment of the nature of the differences between stable bilayers and those that readily convert into inverted phases. Such studies are important because these differences in physical properties often lead to altered membrane functional behavior.

Some aspects of reactions of benzyl isothiocyanate with bovine sarcoplasmic proteins

Benzyl-ITC (benzyl isothiocyanate) reacts preferentially with amino groups and sulfhydryl side chains of bovine sarcoplasmic proteins to form thiourea and dithiocarbamate derivatives, generally resulting in a decrease in solubility of the derivatives along a wide pH range. Under these conditions, it was also possible to show that secondary amine side chains, as found in tryptophan, also react with benzyl-ITC. A quenching of tryptophan fluorescence intensity after interaction with benzyl-ITC was also observed. Polyacrylamide gel electrophoresis (PAGE) experiments in the presence of urea indicated changes in electrophoretical mobility and in composition of subfractions. Changes in surface hydrophobicity and composition as determined by the ANS (8-anilinonaphthalene-1-sulfonate) method and RP-HPLC were also observed. Polymerisation of protein molecules after reaction with benzyl-ITC was documented using the SDS-PAGE technique. These investigations showed that a group of subfractions belonging mainly to glycolytic enzymes and associated proteins with molecular weight between 38-70 kDa was highly reactive.