Partial Protein Denaturation Research Articles

Ultra-High Pressure Homogenization enhances physicochemical properties of soy protein isolate-stabilized emulsions

The effect of Ultra-High Pressure Homogenization (UHPH, 100–300MPa) on the physicochemical properties of oil-in-water emulsions prepared with 4.0% (w/v) of soy protein isolate (SPI) and soybean oil (10 and 20%, v/v) was studied and compared to emulsions treated by conventional homogenization (CH, 15MPa). CH emulsions were prepared with non-heated and heated (95°C for 15min) SPI dispersions. Emulsions were characterized by particle size determination with laser diffraction, rheological properties using a rotational rheometer by applying measurements of flow curve and by transmission electron microscopy. The variation on particle size and creaming was assessed by Turbiscan® analysis, and visual observation of the emulsions was also carried out. UHPH emulsions showed much smaller d3.2 values and greater physical stability than CH emulsions. The thermal treatment of SPI prior CH process did not improve physical stability properties. In addition, emulsions containing 20% of oil exhibited greater physical stability compared to emulsions containing 10% of oil. Particularly, UHPH emulsions treated at 100 and 200MPa with 20% of oil were the most stable due to low particle size values (d3.2 and Span), greater viscosity and partial protein denaturation. These results address the physical stability improvement of protein isolate-stabilized emulsions by using the emerging UHPH technology.

The influence of thermal processing on emulsion properties of defatted hazelnut flour

In this study, the influences of roasting and the amount of hazelnut flour on the formation and stabilization of emulsions containing different amounts of oil were investigated. After hazelnuts were roasted in an oven at 140°C for 40min, the oil content was removed. The emulsions with defatted hazelnut flour containing corn oil at 3%, 10% and 50% were prepared. Roasting process significantly decreased the interfacial tension values of samples down to 1.9mN/m due to protein denaturation. There was no significant difference between the particle sizes of oil droplets in emulsions with roasted and raw hazelnut flour at the same concentration. However, diffusion coefficients of oil droplets increased for emulsions containing roasted defatted flour samples. The zeta (ζ) potential values of all emulsions increased when roasted hazelnut flour was used, indicating the stabilization of suspensions and the solution resistance against aggregation. Storage modulus (G′), loss modulus (G″) and complex viscosity (η∗) of emulsions were measured. G′ value was found to be greater than the G″ value, which fits into weak gel model. The roasting process resulted with lower transition temperatures but with increased transition enthalpies of the flour samples based on differential scanning (DSC) measurements. Lower transition temperatures may be attributed to the partial gelatinization of starch in the flour and partial denaturation of proteins. These results may help to tailor the properties of defatted hazelnut flour when it is used in food products containing emulsions such as sauces, dressings and creams for stabilizing purposes.

Stable Amyloid Oligomers can Seed Fibril Growth Near Physiological Conditions

Spontaneous formation and deposition of protein fibrils with cross beta-sheet structure are associated with an increasing number of human disorders including Alzheimer's disease, senile systemic or dialysis-related amyloidosis. The morphological and structural similarities of amyloid deposits and their various intermediates suggest that amyloid diseases share basic aspects of their etiology. Elucidating the molecular mechanisms underlying these similarities has been challenging since biophysical studies of fibril formation in vitro require partial denaturation of native proteins. These conditions are far from the physiological environment relevant to fibril growth in vitro. Here we show that metastable amyloid oligomers and protofibrils of lysozyme, transiently formed under denaturing conditions, can become stabilized at physiological conditions. In addition, they are capable of inducing the conversion of natively folded, monomeric lysozyme into aggregated β-sheet rich fibrils in vitro. There was no discernible threshold concentration of lysozyme monomers below which seeding didn't induce growth.Our data suggest that only the nucleation of amyloid oligomers is a low-probability event requiring partially denaturing conditions. Once formed, these seeds can become stabilized under near physiological conditions and induce seed elongation and fragmentation. The stabilization of amyloidogenic intermediates away from nucleation conditions, their ability to remain soluble or to seed subsequent fibril growth of native monomers appear to be protein-specific. The oligomer/protofibril-induced growth of native lysozyme observed in our experiments bears intriguing similarities with the ability of prion proteins to drive the autocatalytic conversion of their native counterpart into amyloid fibrils.

FTIR Imaging for Structural Analysis of Frankfurter Sausages Subjected to Salt Reduction and Salt Substitution

In this study, the effects of NaCl, KCl, and MgSO4 in various concentrations on structural and sensory properties of frankfurter sausages were investigated. FTIR was used to analyze the overall homogeneousness of the sausages by simultaneously following the distribution of main sausage ingredients, i.e., proteins, fats, and starch. A more homogeneous distribution of the main ingredients was observed with higher concentration of added salts, while it was most pronounced for the MgSO4 recipe. Furthermore, FTIR imaging was used in order to follow the distribution of protein secondary structure motifs throughout the sausage matrix. It was confirmed that KCl inhibited the partial denaturation of proteins, unlike that observed for MgSO4 recipes, where an additional increase in protein hydration was detected. These findings were unequivocally supported by WHC measurements. However, the sensory analysis clearly distinguished the sausages prepared with MgSO4 due to undesired sensory attributes, which underlines the necessity for using taste masking agents.

Reversibility of Membrane N-Glycome of HeLa Cells upon Treatment with Epigenetic Inhibitors

Glycans are essential regulators of protein function and are now in the focus of research in many physiological and pathophysiological processes. There are numerous modes of regulating their biosynthesis, including epigenetic mechanisms implicated in the expression of glyco-genes. Since N-glycans located at the cell membrane define intercellular communication as well as a cellular response to a given environment, we developed a method to preferentially analyze this fraction of glycans. The method is based on incorporation of living cells into polyacrylamide gels, partial denaturation of membrane proteins with 3 M urea and subsequent release of N-glycans with PNGase F followed by HPLC analysis. Using this newly developed method, we revealed multiple effects of epigenetic inhibitors Trichostatin A, sodium butyrate and zebularine on the composition of N-glycans in human cells. The induced changes were found to be reversible after inhibitor removal. Given that many epigenetic inhibitors are currently explored as a therapeutic strategy in treatment of cancer, wherein surface glycans play an important role, the presented work contributes to our understanding of their efficiency in altering the N-glycan profile of cancer cells in culture.

Effects of high hydrostatic pressure on physicochemical and functional properties of walnut (Juglans regia L.) protein isolate

Walnut (Juglans regia L.) is a good source of protein that has potential application in new product formation and fortification. The main objectives of this study were to investigate the effects of high hydrostatic pressure (HHP) treatment (300-600 MPa 20 min) on physicochemical and functional properties of walnut protein isolate (WPI) using various analytical techniques at room temperature. The results showed significant modification of solubility, free sulfhydryl content and surface hydrophobicity with increased levels of HHP treatment, indicating partial denaturation and aggregation of proteins. Differential scanning calorimetry and fluorescence spectrum analyses demonstrated that HHP treatment resulted in gradual unfolding of protein structure. Emulsifying activity index was significantly (P < 0.05) increased after HHP treatment at 400 MPa, but significantly decreased (P < 0.05) relative to the untreated WPI with further increase in pressure. HHP treatment at 300-600 MPa significantly decreased emulsion stability index. Additionally, HHP-treated walnut proteins showed better foaming properties and in vitro digestibility. These results suggest that HHP treatment could be applied to modify the properties of walnut proteins by appropriate of pressure levels, which will help in using walnut protein as a potential food ingredient.

Effects of wet heat treatment on the germination of individual spores of Clostridium perfringens

To analyse the effect of wet heat treatment on nutrient and non-nutrient germination of individual spores of Clostridium perfringens. Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the dynamic germination of individual untreated and wet heat-treated spores of Cl.perfringens with various germinants. When incubated in water at 90-100°C for 10-30min, more than 90% of spores were inactivated but 50-80% retained their Ca(2+) -dipicolinic acid (CaDPA). The wet heat-treated spores that lost CaDPA exhibited extensive protein denaturation as seen in the 1640-1680cm(-1) (amide I) and 1230-1340cm(-1) (amide III) regions of Raman spectra, while spores that retained CaDPA showed partial protein denaturation. Wet heat-treated spores that retained CaDPA germinated with KCl or l-asparagine, but wet heat treatment increased values of T(lag) , ΔT(release) and ΔT(lys) , during which spores initiated release of the majority of their CaDPA after mixing with germinant, released >90% of their CaDPA and completed the decrease in their DIC intensity because of cortex hydrolysis, respectively. Untreated Cl.perfringens spores lacking the essential cortex-lytic enzyme (CLE), SleC, exhibited longer T(lag) and ΔT(release) values during KCl germination than wild-type spores and germinated poorly with CaDPA. Wet heat-treated wild-type spores germinating with CaDPA or dodecylamine exhibited increased T(lag) , ΔT(release) and ΔT(lys) values, as did wet heat-treated sleC spores germinating with dodecylamine. (i) Some proteins important in Cl.perfringens spore germination are damaged by wet heat treatment; (ii) the CLE SleC or the serine protease CspB that activates SleC might be germination proteins damaged by wet heat; and (iii) the CaDPA release process seems likely to be damaged by wet heat. This study provides information on the germination of individual Cl.perfringens spores and improves the understanding of effects of wet heat treatment on spores.

Occurrence and Characterization of Pale, Soft, Exudative-Like Broiler Muscle Commercially Produced in China

Pale, soft, exudative-like (PSE-like) broiler muscle is a growing problem for meat industry all over the world. However, limited studies have been made to assess broiler meat quality in China. The aim of this study was to investigate the characteristics and incidence of PSE-like broiler muscle commercially produced in China. A total of 1 274 Pectoralis muscles of Arbor Acre broiler were randomly obtained from the processing line to determine the commercial incidence of PSE-like muscle based on color. Furthermore, broiler Pectoralis muscles selected from the 1 274 muscle samples were classified as PSE-like muscle (L*>53, n=33) and normal muscle (L*>48 and L*=53, n=33) to assess meat quality. It was determined that PSE-like muscle had lower muscle pH values, lower water-holding capacity (WHC), lower sarcoplasmic protein solubility, and lower total protein solubility than the normal muscle did. SDS-PAGE profile also showed that bands of approximate 96 and 24 kDa in sarcoplasmic protein and myofibrillar protein varied between these two groups, suggesting partial denaturation of sarcoplasmic proteins and precipitation on myofillarments. Correlation analysis showed that L* values have significant correlation with WHC and protein solubility. Furthermore, the distribution of L* values exhibited a normal curve with range varying from 42.70 to 58.37. It was considered that approximately 23.39% of the population was PSE-like muscle. These results suggest that PSE-like meat can represent a significant portion of commercially processed broiler breast meat in China and that the L* value measurement could be used to sort broiler meat quality using a cut-off point.

Development and validation of a rapid high-performance liquid chromatography-tandem mass spectrometry method for the determination of WJ-38, a novel aldose reductase inhibitor, in rat plasma and its application to a pharmacokinetic study

WJ-38 is an aldose reductase inhibitor that is being developed for the treatment of diabetic complications. The present paper describes a sensitive and specific liquid chromatography-tandem mass spectrometry method for the determination of WJ-38 in rat plasma. Partial denaturation of plasma proteins with methanol followed by liquid–liquid extraction using ethyl acetate was used to extract strongly protein-bound WJ-38 from rat plasma. Chromatographic separation was performed on an Inertsil ODS-3 column with an isocratic mobile phase consisting of acetonitrile, water and formic acid (75:25:0.125, v/v/v). Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an ESI interface operating in positive ionization mode. Quantitation was performed using selected reaction monitoring of precursor-product ion transitions at m/z 392→246 for WJ-38 and m/z 446→321 for glipizide (internal standard). A linear calibration curve was obtained over the concentration range of 10.0–10,000ng/mL for WJ-38 in rat plasma. The intra- and inter-day precisions were less than 13.6% and the accuracy was within ±5.3%. The extraction recovery of WJ-38 from rat plasma was over 66.0%. The validated method has been successfully applied to a pharmacokinetic study in rats after intragastrical administration of WJ-38.

The impact of protein disulfide bonds on the amyloid fibril morphology

Amyloid fibrils are associated with many neurodegenerative diseases. Being formed from more than 20 different proteins that are functionally or structurally unrelated, amyloid fibrils share a common cross-β core structure. It is a well-accepted hypothesis that fibril biological activity and the associated toxicity vary with their morphology. Partial denaturation of a native protein usually precedes the initial stage of fibrillation, namely the nucleation process. Low pH and elevated temperature, typical conditions of amyloid fibril formation in vitro, resulted in partial denaturation of the proteins. Cleavage of disulfide bonds results typically in significant disruption of protein native structure and in the formation of the molten global state. Herein we report on a comparative investigation of fibril formation by apo-α-lactalbumin and its analog that contains only one of the four original disulfide bonds using deep UV resonance and non-resonance Raman spectroscopy and atomic force microscopy. Significant differences in the aggregation mechanism and the resulting fibril morphology were found.

Adsorption of antibody onto Pluronic F68-covered nanoparticles: link with surface properties

The use of nanoparticles as drug delivery systems is an emerging application to improve intravenous therapy. Controlling the biocompatibility of the nanoparticles is a crucial step towards the optimal implementation of these systems. Adsorption of serum components onto the nanoparticles is driven mainly by hydrophobic forces. Thus, incorporation of hydrophilic polymers such as polyethylene oxide (PEO) derivatives to the nanostructure surface reduces the interaction of nanoparticles with blood stream components (IgG). The effectiveness of the poloxamer for reducing protein adsorption depends on the resistance of this coating layer. A fundamental understanding of the properties of this surface coating is crucial towards the rational design of these systems. Here, we have used an innovative combination of experimental techniques to evaluate the properties of the nanoparticles and more specifically, the mechanical properties of the coating. Electrophoretic mobility and colloidal stability data suggest similar surface characteristics between IgG–Pluronic–polystyrene (PS) and IgG–PS complexes, indicating that the protein adsorption is just slightly reduced by the presence of poloxamer. Nevertheless, the biological activity of the adhered antibodies suggests that the Pluronic F68 significantly altered their immunoactivity. The decrease in the activity might indicate a partial denaturation of the protein and/or changes in the preferential orientation when adsorbing caused by the surfactant–protein interactions. The surface characterisation of the IgG layers adsorbed onto a Pluronic covered surface importantly provides evidence of the conformational change undergone by the protein, supporting the partial protein denaturation suggested by the loss of immunoreactivity in the IgG–Pluronic–PS particles. The use of surface tension to obtain structural and mechanical information about the coating procedure is a novel approach to understand generic features of the biocompatibility of colloidal systems. These results may help to understand why drug nanocarriers coated by poloxamers improve their long-circulating properties in comparison with uncoated particles.

Influence of pH on Structure and Function of Amaranth (Amaranthus hypochondriacus) Protein Isolates

ABSTRACTStructural and functional properties of two amaranth protein isolates as a function of pH were studied. Isolates, A9 and A11, were obtained by alkaline extraction at pH 9 and 11, respectively. Gel filtration chromatograms of A9 and A11 showed similar profiles. The A11 isolate contained mainly albumins and globulins, and a small proportion of globulin‐P aggregates, suggesting the presence of species with a higher degree of denaturation compared to A9. Differential scanning calorimetry (DSC) showed that A9 was characterized by two thermal transitions (65.8 and 98°C); A11 exhibited only a small endotherm (66.6°C) and a second, less defined one. DSC analysis of A9 at pH 2–4 did not show endotherms, but at pH 5, some protein structures were observed. A11 showed a greater degree of denaturation. FPLC results showed that the proteins in A9 are more folded and their conformation is closer to the native state than those in A11, which are more unfolded due to pH‐mediated denaturation, mainly in acid media. The surface hydrophobicity of the isolates in acid media was lower than in alkaline media. The fluorescence emission spectra of the isolates showed differences in acidic pH conditions. As expected, the highest solubility was at alkaline pH. The water‐holding capacity was similar for both isolates. The water‐imbibing capacity and speed of foaming was higher for A11 than for A9. In summary, intense pH treatment of amaranth isolates generated partial or total protein denaturation and differences in the functional properties.

Characterization of single heat-activated Bacillus spores using laser tweezers Raman spectroscopy

Heat activation of dormant bacterial spores is a short treatment at a sublethal temperature that potentiates and synchronizes spore germination. In this paper, laser tweezers Raman spectroscopy (LTRS) was used to study the heat activation of single spores of Bacillus cereus and Bacillus subtilis. We measured the Raman spectra of single spores without treatment, during heat activation at 65 degrees C (B. cereus) or 70 degrees C (B. subtilis), and following heat activation and cooling to 25 degrees C. Principle component analysis (PCA) was applied to discriminate among the three groups of spores based on their Raman spectra. The results indicated that: (1) there are large changes in the Raman bands of Ca-DPA and protein for both B. cereus and B. subtilis spores during heat activation, indicative of changes in spore core state and partial protein denaturation at the heat activation temperatures; (2) these spectral changes become smaller once the heated spores are cooled, consistent with heat activation being reversible; (3) minor spectral differences between untreated and heat-activated and cooled spores can be discriminated by PCA based on non-polarized and polarized Raman spectra; and (4) analysis based on polarized Raman spectra reveals that partial denaturation of protein during heat activation is mainly observed in the vertically polarized component.

Generating substrate bound functional chemokine gradients in vitro

Microcontact printing (mCP) is employed to generate discontinuous microscale gradients of active fractalkine, a chemokine expressed by endothelial cells near sites of inflammation where it is believed to form concentration gradients descending away from the inflamed area. In vivo, fractalkine is a transmembrane molecule extending its chemokine domain into the vascular lumen. Substrate bound in vitro gradients may thus closely resemble in vivo conditions. Direct mCP of sensitive proteins like fractalkine may cause partial protein denaturation and will not ensure correct orientation of the biologically active part of the molecules. Here, indirect mCP of a capture antibody recognizing a molecular tag on the target protein is successfully used to pattern tagged fractalkine in microscale gradient patterns. Fractalkine functions as an adhesion molecule for leukocytes. Cells expressing the fractalkine receptor are found to attach to the gradient structure at a density correlated with the fractional area covered by fractalkine. This indicates that the patterned fractalkine maintains its biological function. The method can be applied to in vitro studies of cell responses to the wide range of naturally surface-bound chemokines (haptotactic gradients). The use of a capture antibody facilitates control of the orientation of tagged molecules, thereby ensuring a high degree of bio-functionality through correct presentation and reduced protein denaturation.

Characterization of the Electron Transfer of a Ferrocene Redox Probe and a Histidine-Tagged Hemoprotein Specifically Bound to a Nitrilotriacetic-Terminated Self-Assembled Monolayer

We report the selective, controlled binding of a model redox probe, 1,1'-bis(N-imidazolylmethyl)ferrocene (Fc-Im2), and a small redox hemoprotein, histidine-tagged recombinant human neuroglobin (hNb), at the surface of metal electrodes (gold and SER-active silver) modified by a self-assembled monolayer (SAM) of a nitrilotriacetic (NTA)-terminated thiol. The resulting SAMs were characterized by cyclic voltammetry and surface-enhanced resonance Raman (SERR) spectroscopy coupled to electrochemistry. Once specifically bounded to the Ni(II)-NTA-modified gold electrode, nearly ideal cyclic voltammetric behavior with relatively fast electron-transfer (ET) communication through the SAM was determined for the Fc-Im2 redox probe. However, no direct electron transfer could be evidenced for the hNb redox protein under the same conditions. This outcome was different from the result obtained during SERR experiments coupled to electrochemistry in which a direct electrochemical conversion of hNb immobilized on a Ni(II)-NTA-modified SER-active Ag electrode was observed. The SERR spectra of the immobilized hNb was the same as the resonance Raman spectra of the protein in homogeneous solution, allowing us to conclude that the native structure of hNb was retained upon immobilization and that the direct ET was not the result of some partial or complete protein denaturation. The long-range ET rate constant (kET) through the SAM was determined by time-resolved SERR spectroscopy. A value of kET=0.12 s(-1) was obtained, which is within the predicted range of a fully nonadiabatic ET through a SAM thickness of approximately 26 A and close to the values previously determined for analogous small redox proteins at similar long-range ET distances. A SERR spectroelectrochemical titration of the immobilized hNb was also carried out, showing both an apparent standard potential (E0') negatively shifted by 100 mV compared with hNb in solution and a gentle slope in the titration curve. These results suggest a range of chemical environments in the surroundings of the redox protein and a variety of interactions with the NTA-terminated SAM. The influence of protein immobilization on E0' is discussed together with the long-range ET rate constant and molecular orientation of the surface-immobilized hNb.

Foam fractionation of α‐lactalbumin and β‐lactoglobulin from a whey solution

AbstractWhey, a byproduct of cheese production, is often considered a waste stream. Separation and purification of whey proteins is a difficult and expensive task. Occasionally, it is transported out of the dairy plant for a per volume charge. One possible method to reduce the waste volume and disposal cost is to concentrate whey by foam fractionation and potentially produce a valuable coproduct, a concentrated whey protein solution. Foam fractionation is an adsorptive bubble separation method based on the hydrophobic/hydrophilic properties of proteins. In this study, foam fractionation was evaluated for the concentration of whey proteins, specifically α‐lactalbumin and β‐lactoglobulin, from a dilute whey protein solution. The effects of initial whey protein concentration (0.075 and 0.15 mg/ml), pH (3.8–5.5), superficial gas velocity (0.85 and 0.95 cm/s) and temperature (4 and 65 °C) on protein enrichment and recovery were examined. Higher enrichment was achieved with the lower initial protein concentration (0.075 mg/ml), and at pH values that were near the isoelectric points (pI) of α‐lactalbumin and β‐lactoglobulin (pH 3.8, 4.2, 4.5, and 5.2). Higher superficial gas velocity enhanced the amount of proteins recovered with a decrease in the enrichment. Cold temperature treatment and partial heat denaturation of whey proteins reduced enrichment and increased protein recovery simultaneously. Copyright © 2009 Curtin University of Technology and John Wiley &amp; Sons, Ltd.

Calorimetric study of extracellular tissue matrix degradation and instability after gamma irradiation

Native extracellular tissue matrix (ECM) is increasingly used for tissue repair and regeneration. The kinetics of gamma irradiation damage on human dermis ECM was studied by differential scanning calorimetry (DSC). Dermis ECM was irradiated at a low-dose rate of 0.23 kGy h −1 in order to study the progression of ECM damage as the gamma dose increased from 0 to 32 kGy. The study showed that the effect of gamma irradiation above 2 kGy was predominantly peptide chain scission. As the gamma dose increased, the stability of irradiated ECM decreased further, and multiple ECM domains of different stability were detected. Even a moderate gamma dose (7–12 kGy) could decrease the onset denaturation temperature of ECM to below body temperature. DSC analysis also showed partial and spontaneous protein denaturation in gamma-irradiated, rehydrated ECM at 37 °C. In vitro rehydration tests confirmed that a significant fraction of the irradiated ECM disintegrated into minute ECM fragments at 37 °C, although the irradiated ECM appeared to be normal at 4 °C and room temperature. DSC data were correlated well to effects of gamma irradiation on ECM microstructure, mechanical property and in vitro cell response reported earlier by us. A model was presented to describe the kinetics of gamma-irradiation-induced alterations of tissue ECM properties.

Site-Specific Pegylation of G-CSF by Reversible Denaturation

A new strategy has been developed for extending the possibility of poly(ethylene glycol) (PEG) modification to accessible thiol groups of biologically active proteins. In particular, thiol-reactive PEGs have been coupled to the cysteine 17 of granulocyte colony stimulating factor (G-CSF), which is known to be partially buried in a hydrophobic protein pocket. The PEG linking was accomplished by partial protein denaturation with 3 M guanidine.HCl in the absence of any reducing agent in order to preserve the native protein's disulfide bridges. PEG coupling occurred also, but at a lower degree, by using a 3 M solution of urea as the denaturing agent. Following the PEGylation, which was carried out in the unfolded state, the conjugated protein was refolded using dialysis or gel filtration chromatography to eliminate the denaturant. Different thiol-reactive PEGs and polymer molecular weights (5, 10, or 20 kDa) were investigated for G-CSF conjugation under denaturation. The secondary structure of the protein in the G-CSF-PEG conjugates, evaluated using circular dichroism and biological activity assay in cell culture, was maintained with respect to the native protein. Unexpectedly, conjugation enhanced the G-CSF tendency to aggregate, a problem that was overcome by a proper formulation.

Oxidative stress-mediated protein conformation changes: ESR study of spin-labelledstaphylococcal nuclease

We report on the electron spin resonance (ESR) study of the photo-oxidative stress-mediatedprotein conformation changes in the spin-labelled protein staphylococcal nuclease (SNase).The photo-oxidative stress was brought on by photosensitization of singlet oxygen(1Δg) in the presence of a novel photosensitizer, water-soluble fullerolC60(OH)19(ONa)17, and resulted in partial protein denaturation. This process was monitored via ESRmeasurements performed for a spin-labelled SNase Thr-62-Cys mutant, with MTSSL spin label(SL) attached to the cysteine 62 residue (SNase T62C-SL). Prior to ESR measurements ofthe oxidative stress-induced alterations in protein conformations, the efficiency ofC60(OH)19(ONa)17 for 1Δg-generation was confirmed by three different techniques: (i) selective reactive scavenging of1Δg and ESR detection of the resulting paramagnetic product, (ii)1Δg-mediated photo-oxidative loss of tryptophan and (iii) by measuring the characteristicnear-infrared phosphorescence of singlet oxygen at 1270 nm. The observed evolution of theESR spectra of SNase T62C-SL as a function of exposure to the photo-oxidative stresspoints to marked changes in protein conformation due to the deleterious action of1Δg.

EFFECT OF MICROWAVES ON THE NITROGEN DISTRIBUTION AND SOME PHYSICAL PROPERTIES AND MICROBIAL CONTENT ON SHEEPS MILK

Sheep milk was subjected to microwave for 60-180 sec., and analysed for nitrogen distribution , pH and rennet coagulation time.Also analysed for total bacterial count,Co- liform bacteria ,Staphylococcus and Salmonella. Heating with microwaves improved the bacteriological quality of milk ,but resulted in partial denaturation of whey proteins ,and Increased the ratio of casein nitrogen and non protein nitrogen,and retardation of the rennet coagulation time of milk.