An approach to apply surface hydrophobicity changes as an indicator of lysozyme modification

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1 An approach to apply surface hydrophobicity changes as an indicator of lysozyme modification G. LESNIEROWSKI*, P. KONIECZNY and J. KIJOWSKI Department of Food Quality Management, August Cieszkowski Agricultural University, Wojska Polskiego 31, PL Poznan, Poland Keywords: lysozyme; modification; surface hydrophobicity; detergent binding method Summary Lysozyme is an enzyme, which is found widespread in nature. Lysozyme occurs mostly in the natural environment as a monomer, which breaks down cell walls of Gram-positive bacteria. Formation of polymeric forms resulting from the modification of the enzyme increases its action against Gramnegative bacteria without any loss of its antibacterial activity. Since modifications lead to changes of enzyme properties, the objective evaluation of modified lysozyme is indeed a difficult task. Currently, methods basing on principles of spectrophotometry, electrophoresis and differential scanning calorimetry (DSC) are used for this purpose. None of these methods makes it possible to determine changes in the hydrophobic surface of the enzyme resulting from its modifications. The aim of this study was to characterize changes of hydrophobic surface properties of modified lysozyme in comparison to its unmodified monomer form. To determine their surface hydrophobicity, the interference of dye binding in a protein assay (BIO-RAD Dye Reagent) due to the covering of hydrophobic sites of the protein with Tween 80 (polyoxyethylene sodium monooleate) was measured. The effect of both the dye dilution, and detergent and lysozyme concentrations on the surface hydrophobicity of examined proteins was taken into consideration. As indicated by the performed experiments, apart from the factors mentioned above, the procedure for the preparation of lysozyme was essential for the obtained results. The following analytical parameters will be recommended for the determination of surface hydrophobicity of lysozyme with the examined method: dye dilution of 1 : 50, detergent concentration of 0.25 % and protein concentration of 0.25 %. Data collected in this study have shown that in comparison to the monomer form the hydrophobic surface of modified lysozyme changed markedly with the increase in the number of polymeric forms in the examined preparations. Further investigations in this respect will be recommended before the formulation of final conclusions. Introduction The modification of lysozyme leads to the changes of its hydrophobic properties. A characteristic feature of the enzyme molecule is its nonpolar interior. During modification, especially when thermal treatment methods are used, hydrophobic amino acid residues are exposed outside of the molecule. Changes of hydrophobicity are also associated with changes of antibacterial properties of lysozyme. The modified enzyme exhibits markedly higher antibacterial activity, particularly against Gramnegative bacteria. Therefore, it is extremely important to determine as accurate as possible the degree of changes occurred during the lysozyme modification. The measurement and comparison of surface hydrophobicities for non modified versus modified form of lysozyme seems to be a proper research method in this respect. According to the references review, this approach was not performed yet, possibly due to the expected instrumental as well as methodological difficulties. Among the methods available for determination of surface protein hydrophobicity, the majority of them represent advanced instrumental and time consuming techniques, where special equipment and reagents are required. Additionally, some of these methods are useful for soluble proteins only (Nakai et al., 1996, Konieczny, 2001). However measurement of surface hydrophobicity is still quite controversial so that no standard method has ever been established, for routine determination of protein hydrophobicity less time- and labourconsuming method is preferred. The method elaborated by Lieske and Konrad, 1994 and applied in this study fulfills all criteria mentioned above. The aim of this study was, therefore, to characterize 207

2 using this method the changes of hydrophobic surface properties of modified lysozyme versus its unmodified monomer form. Materials and methods MATERIALS The experimental material was the monomer of hen egg white lysozyme isolated using the chromatographic method with the application of an ion exchanger (Lesnierowski, 1997), lysozyme dimer (Lydium KLP by Nika) and preparations of lysozyme modified thermally, chemically, thermochemically and using the membrane method. Enzyme modifications were performed according to the methodologies developed at the Department of Food Quality Management, Agricultural University of Poznan. DETERMINATION OF LYSOZYME HYDROLYTIC ACTIVITY Lysozyme hydrolytic activity was determined with the use of spectrophotometry, the principle of which is based on the phenomenon of cell wall lysis in Micrococcus lysodeikticus bacteria by the enzyme (Lesnierowski and Kijowski, 1995). The lytic activity of lysozyme was determined by monitoring the decrease in turbility of a suspension of Micrococcus lysodeikticus cells at 450 nm. The activity was presented as the rate of decrease in absorbance per min of the initial velocity of reaction ( abs/min). ELECTROPHORESIS Electrophoretic analysis was conducted on polyacrylamide gel using the SE-600 apparatus (Hoefer Scientific Instruments). Electrophoresis in SDS-PAGE was carried out according to the method of Laemmli (1970) and Lesnierowski (1997) using 12.5% acrylamide separating and 6% stacking gels containing 0.1% SDS. Electrophoresis was run at the current of 60 ma in stacking gel and 90 ma in separating gel for about 5 hours using an electrophoretic buffer of Tris-glycine containing 0.1% SDS. The gels were stained with 0.025% Coomassie Brilliant Blue R solution. The following were used as standards: Lysozyme 14.6 kda (Sigma), Lydium KLP 28 kda (Nika Health Product) and Hen Albumen 45 kda (Sigma). The amounts of polymers in individual samples were calculated with the application of the TotalLab v.2.0 software. DETERMINATION OF CHANGES IN HYDROPHOBIC SURFACE Surface hydrophobicity of lysozyme preparations was determined using polyoxyethylene sodium monooleate (Tween 80, SERVA, Germany) as a ligand. The interference of dye binding in a protein assay (BIO-RAD Dye Reagent No ) due to the covering of hydrophobic sites of the protein with the detergent was measured spectrophotometrically. Reagent and sample preparation, as well as the assay procedure were carried out with slight modifications as described by Lieske and Konrad, Surface hydrophobicity of the protein was calculated from the results of 2 different protein assays, which were developed at the same time. (1a) 50 µl protein solution (lysozyme preparation in 0.05 M phosphate buffer, ph 6.5 ) prepared previously in the sonicator (UM20 type, 25 khz) were placed on the bottom of clean and dry test tubes, whereas 50 µl buffer solution were used as blank. (1b) The same procedure as (1a), but with the addition of 50 µl detergent (Tween 80) solution in each tube. (2). Detergent containing tubes (1b) were shaken in a sonicator for at least 5 min at C, to complete thorough protein and detergent binding (3) 2.5 ml diluted dye reagent (Bio RAD in aqua dest.) were added to each tube and shaken carefully without foaming. (4) All tubes were allowed to stand for 12 min to develop the colour. (5) Colour intensity of each separate tube [a, b, a, b ] was measured at λ= 595 nm vs M phosphate buffer, ph

3 Protein hydrophobicity (PH) was defined as follows: PH [ nonpolar residues] = [ a b ] [ a b ] = [ nonpolar residues] + [polar residues] [ a b] 100 Changes in surface hydrophobicity of nonmodified (monomer) vs. modified forms of lysozyme protein were expressed as relative values ( PH). STATISTICAL ANALYSIS The obtained results of the determination were subjected to statistical analysis using STATISTICA v.6.0 software. Results and discussion CHARACTERISTIC OF SELECTED PROPERTIES OF INVESTIGATED LYSOZYME PREPARATIONS The following lysozyme preparations were studied: T- lysozyme modified thermally, Ch - lysozyme modified chemically, Tch- lysozyme modified thermochemically, M- lysozyme modified by membrane techniques. Selected properties of the tested preparations are presented in Table 1. Table 1 Characteristic of selected properties of investigated lysozyme preparations. Parameter Lysozyme preparations Monomer T Ch Tch M Dimer Percentage of monomer Percentage of polymers: dimer trimer total Hydrolytic activity (U/mg) Further research included the evaluation of the effect of dye, detergent and protein concentrations on changes in hydrophobic surface of modified lysozyme in comparison to its nonmodified form (monomer). THE EFFECT OF DYE CONCENTRATION ON DETERMINATIONS OF HYDROPHOBICITY CHANGES IN MODIFIED LYSOZYME The dye reagent by Bio-RAD, which are commercially available as a concentrate, was diluted many times in order to find the range of concentrations making it possible to measure absorbance of solutions of all the tested lysozyme preparations under identical analytical conditions. It was determined in preliminary investigations that at the dilution of the dye not higher than 20 times the coloration of lysozyme solutions was too intense to depict differences between absorbance values of lysozyme monomer and modified forms. Only further dilution of the dye (1:25, 1:50, 1:100) made it possible to characterize the direction of changes in the hydrophobic surface of the tested lysozyme preparations, as presented in Table 2. It turned out that along with the increase in the percentage of polymeric lysozyme forms an increase in PH values was observed, for each dye dilution varied 209

4 values were obtained for individual lysozyme preparations, and for the most part the differences between the investigated samples were statistically significant (p< 0.05). It was observed that at the dye dilution of 1:25 the obtained results were distinctly lower than for the other dilutions and samples T and Tch did not differ significantly (p<0.05). These differences were statistically significant (p<0.05) for all the tested samples only at the successive dye dilutions, i.e. 1:50. The obtained results showed (Table 2) that in order to determine hydrophobicity changes in modified lysozyme using the method described above solutions with either too low or too high dye dilutions may not be used. It was found that the assay should optimally be done at dye dilution of 1:50. Table 2 Changes in the hydrophobic surface PH in preparations of modified lysozyme depending on the dilution of the dye (Bio-RAD). Sample Percentage of Dye dilution PH polymeric forms Monomer 0 1: a T 27 1: b Ch 33 1: c Tch 45 1: c M 52 1: d Dimer 100 1: i Monomer 0 1: a T 27 1: c Ch 33 1: e Tch 45 1: f M 52 1: h Dimer 100 1: l Monomer 0 1: a T 27 1: b Ch 33 1: d Tch 45 1: f M 52 1: g Dimer 100 1: k a-k identical letters at values denote a lack of significant differences at p<0.05. THE EFFECT OF DETERGENT CONCENTRATION ON THE DETERMINATION OF HYDROPHOBICITY CHANGES IN MODIFIED LYSOZYME Another investigated variability factor was the concentration of the detergent, which was used for assays in this study at the range from 0.20 to 0.30%. Detergent binding and all the related aspects play the key role in the assessment of surface hydrophobicity according to the proposed method as indicated previously (Lieske and Konrad, 1994, Lieske and Konrad, 1995). The results of investigations on changes in hydrophobic surface PH of modified lysozyme in comparison to its monomer (nonmodified form) for various concentrations of detergent solutions are presented in Table 3. In preliminary investigations it was observed that similarly as in case of the determination of the optimum dye concentration, an inappropriately selected range of detergent concentrations made it impossible to interpret the obtained results. As results from Table 3, for concentrations from the selected range, along with the increase in the share of polymorphic forms in the investigated lysozyme preparations, again an upward trend was observed for PH values, and the differences between samples were as a rule statistically significant (p < 0.05). 210

5 Table 3 Changes in hydrophobic surface PH in preparations of modified lysozyme depending on the concentration of detergent solution (Tween 80). Sample Polymer Tween 80 PH Monomer a T c Ch d Tch e M f Dimer i Monomer a T d Ch e Tch g M h Dimer k Monomer a T ab Ch b Tch c M d Dimer h a-k see Table 2 At the detergent concentration of 0.30% for most investigated samples values of limited diversification were obtained, which made the interpretation of the results very difficult. Only at the application of detergent concentration of 0.20% % definitely more diversified results were obtained for individual samples, and at the same time the repeatability of results improved. It needs to be emphasized that the concentration of the detergent Tween 80 of 0.25% was recommended to be applied by the authors of the discussed method of protein hydrophobicity determination (Lieske, Konrad, 1994). The obtained results showed that detergent concentration had a significant effect on the measurement of hydrophobicity changes in the tested protein preparations. It was found that a change in the hydrophobic surface of modified lysozyme needs to be evaluated by the discussed method applying detergent concentrations within the % range. Exceeding this range made it difficult or even impossible to interpret the obtained results. THE EFFECT OF PROTEIN CHANGES ON THE DETERMINATION OF HYDROPHOBICITY CHANGES IN MODIFIED LYSOZYME The content and solubility of protein in the sample is one of the most important factors which need to be taken into account when determining surface hydrophobicity of any protein preparation (Kato and Nakai, 1980, Nakai et al., 1986, Konieczny and Uchman, 2002). The method of determining surface hydrophobicity, proposed by Lieske and Konrad, (1994) and applied in this study, required especially thorough preparation of solutions of lysozyme protein preparations. The precondition for its application was good solubility of the investigated proteins in the selected buffer and obtaining clear solutions for analyses. In this study such an effect was achieved dissolving the tested samples using an ultrasound disintegrator. In this way lysozyme stock solutions were prepared (with protein content of 1.0%), while solutions for direct analyses (with the concentrations of %) were obtained through appropriate dilutions of stock solutions. The results presenting the effect of protein concentration in the solution on changes in the hydrophobic surface PH depending on the type of the preparation and its concentration are shown in Table

6 Table 4 Changes in hydrophobic surface PH in preparations of modified lysozyme depending on protein solution concentration. Sample Polymer Protein PH Monomer a T b Ch d Tch e M f Dimer l Monomer a T c Ch e Tch h M i Dimer l Monomer a T d Ch de Tch g M k Dimer l a-k see Table 2 It was shown that it was possible to determine changes in lysozyme hydrophobicity, similarly as in case of dye and detergent, only in a precisely defined range of protein concentration in the investigated solutions. It was shown that these values fell within the 0.05 to 0.10% range. Too high or too low amounts of lysozyme proteins in the solution (outside the defined range) made it impossible to take measurements and thus to determine the hydrophobicity of the tested samples. The conducted tests showed that hydrophobicity changes in lysozyme solutions may be measured, thus at each protein concentration within the % range, and the best results were obtained for the protein concentration of 0.075%. At the same time it needs to be stressed that the size of changes in hydrophobic surface PH was closely correlated with the enzyme modification rate. Conclusions The conducted investigations showed that the presented method makes it possible to determine changes in hydrophobic surface of modified lysozyme in relation to its monomer form. It turned out that the investigated factors has a significant effect on the final assay results. Precisely defined ranges of protein concentration ( %), detergent concentration ( %) and dye dilution ratio (1:25-1:100) were determined, in which the assay needs to be done. After any of the set range limits was exceeded the obtained results were very difficult or even impossible to interpret. On the other hand, within the established ranges of protein, detergent and dye concentrations various values of changes in hydrophobic surface were obtained for the investigated samples. The following were decided to be the optimal assay conditions: Protein concentration % Detergent concentration 0.25 % Dye dilution 1:50 The presented method of determination of hydrophobicity changes in modified lysozyme is the first such attempt, as so far no such study has been found in literature on the subject. However, it needs to be said that as a relatively simple method, not requiring complex and specialized equipment, it should be performed by researchers with analytical expertise. It pertains especially to the method of sample preparation for assays and precision in the performance of the assay. Finally it may be stated that after meeting rather lenient requirements concerning equipment and personnel, the proposed method may become a routine way to determine lysozyme hydrophobicity. 212

7 References KATO A., MATSUDA T., MATSUDOMI N., KOBAYASHI K. (1984). Determination of protein hydrophobicity using a sodium dodecyl sulfate binding method. J. Agric. Food Chemistry, 32, KATO A., NAKAI S. (1980). Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins. Biochem. Biophys. Acta, 624; KONIECZNY P. (2001). Surface hydrophobicity as determinant factor of selected functional properties of protein preparations. Rocz. AR Pozn. Rozpr. Nauk. 319, (in Polish). KONIECZNY P., UCHMAN W. (2002). Comparative characterization of surface hydrophobicity and other physico-chemical properties of selected protein preparations. EJPAU, Series Food Science and Technology, Vol. 5, Issue 2. LAEMMLI U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, LESNIEROWSKI G. (1997). Obtaining lysozyme from hen egg white using the cristallization, infiltration and ion exchanger methods, Doctoral thesis, Agricultural University of Poznan, (in Polish). LESNIEROWSKI G., KIJOWSKI J. (1995). Enzymatic activity of lysozyme and its application in food preservation. Przemysl Spozywczy, 4/ 95, (in Polish). LIESKE B., KONRAD G. (1994). A new approach to estimate surface hydrophobicity of proteins. Milchwissenschaft, 49: LIESKE B., KONRAD G. (1995). Determination of surface hydrophobicity of milk proteins comparison between a new detergent binding method and hydrophobic interaction FPLC. Milchwissenschaft, 50; (1), NAKAI S., LI CHAN E., ARTEAGA G. (1996). Measurement of surface hydrophobicity. In: Methods of testing protein functionality. Ed. G. M. Hall. Chapmann, London, NAKAI S., E. LI-CHAN, S. HAYAKAWA S. (1986). Contribution of protein hydrophobicity to its functionality, Nahrung, 30,