Binding and Thermodynamic Parameters of Ovalbumin-oleic acid Conjugate. Zeyad Yasseen a* and Baker Zabut b الملخص

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1 Binding and Thermodynamic Parameters of Ovalbumin-oleic acid Conjugate Zeyad Yasseen a* and Baker Zabut b a Assistant Prof. in chemistry, Faculty of Science, Islamic University-Gaza (IUG), Palestine. E-.mail: zyasseen@iugaza.edu.ps. Mobile: Fax: b Associated Prof. in biochemistry, Faculty of Science, Islamic University of Gaza, Palestine. E-.mail: bzabut@iugaza.edu.ps. Mobile: Fax: * corresponding author. ارتباط بروتين بياض البيض بحمض الا وليك و حساب عوامل الديناميكا الحرارية الملخص تهدف الدراسة لتقدير عوامل ارتباط وعوامل الديناميكا الحرارية لعملية اقتران بروتين بياض الب يض م ع حم ض الا ولي ك باستخدام تقنية التوهج الا شعاعي في درجات حرارة مختلفة. تم تحضير المرآب المعق د با ض افة حم ض الا ولي ك إل ى ب روتين بي اض الب يض داخ ل محل ول م نظم فوس فاتي ف ي درج ات حرارة مختلفة و أدت عملية الاقتران هذه إلى تغير في عملية التوهج الا شعاعي للبروتين التي ت م تحليله ا بطريق ة سكتش رد لللا قتران لتقدير عوامل ارتباط المعقد المتكون في درج ات ح رارة ب ين درج ة مي وي ة وت م حس اب مق دار الح رارة الحرة للمعقد من خلال مقدار ثابت الارتباط عند آل درجة حرارة وحساب آل من مقدار المحتوى الحراري ومقدار درجة الفوضى باستخدام معادلة فانت هوف. بين ت النت اي ج أن ثاب ت وع دد مواق ع ارتب اط حم ض الا ولي ك ف ي الب روتين تختل ف ب اختلاف مق دار درج ة الح رارة فك ان متوسط عدد مواق ع الارتب اط ح والي 2.0 و ثاب ت الارتب اط ه و 1 M ف ي درج ة ح رارة 22 مº و متوس ط عدد المواقع آان حوالي 3.0 وثابت الارتباط هو 1 M في درجة حرارة 55 م º وأن عملية تك وين المعق د مصحوبة بانطلاق حرارة ودرجة عالية من الانتظام. ABSTRACT The present study aims to investigate association constants, binding capacity and different thermodynamic parameters of ovalbumin-oleic acid (Oval/OA) conjugate at different temperatures using fluorescence method. Oval/OA conjugate was prepared in a phoshphate buffer solution at different temperatures. The conjugation process resulted in a change of the fluorescence emission intensity. The emission spectra of the conjugate were analyzed according to Scatchard plot to obtain the association constant (k a ) values and the number of binding sites at several temperatures in the range of C. From the K a value, the free energy ( G ) was calculated. The Van t Hoff enthalpy ( H VH ) and entropy ( S ) values associated with the conjugation were estimated using the Van t Hoff equation. The results showed that the binding capacity and the association constant of Oval/OA conjugate were temperature dependent. Average number of binding sites of OA in Oval and K a value at 22 C were about 2.0 and M 1, respectively. On the other hand, at 55 C average OA binding capacity of Oval and K a value were about 3.0 and 0.73 x 10 3 M 1, respectively. The results also showed that the conjugation was enthalpically driven, accompanied by a negative contribution of entropy. Key words: ovalbumin-oleic acid conjugate, fluorescence method, association constants, binding capacity, thermodynamic parameters.

2 INTRODUCTION Proteins interactions with various ligands are very important in explanation of their structure and function. Egg white contains a number of nutrient globular proteins frequently referred to as albumen. Oval is the main protein found in the egg white, making up to 60-65% of the total protein [1]. It belongs to the serpin super family of proteins, although it is unable to inhibit any proteases [2]. It is a phosphorylated glycoprotein of 385 amino acid residues and has a molecular weight of 42.7 KDa. Oval has four sites of postsynthetic modification; in addition to the acetylated N terminus, the carbohydrate moiety is located at Asparagine-292, and the two phosphorylated serines are at residues 68 and 344 [3]. The amino acid sequence of Oval is in complete agreement with the determination of Oval mrna sequence [4]. However, the carbohydrate and phosphate portions account for an additional 1428 and 160 g per mole respectively, giving a total molecular weight of 44.3 KDa [5]. Oval in aqueous solution is readily denatured simply by mechanical agitation and it is found to be resistant to thermal denaturation. However, different scanning calorimetric techniques indicated that oval denatures at 84 C [6]. Oval can be used as a carrier protein to conjugate to synthetic peptides for use as an immunogen. It has the following amino acids: 20 Lysine, 10 Tyrosine, 6 Cystiene, 14 Aspartate, and 33 Glutamate which make it suitable for conjugation [7]. Thus, similar to different sources of albumins, oval is able to conjugate with Cysteine, Glutathione, and numerous hydrophobic ligands including fatty acids [8,9]. However, the coupling of different ligands to Oval represents an advance in the development of oral vaccine [10,11] Conjugation of palmitate to Oval was used successfully to abolish its capacity to induce oral tolerance in experimental animals [12]. Oval chelates to heavy metals and traps the metal ions within the sulfyhdryl bonds of the protein. Chelating prevents the absorption of the metals into the gastrointestinal cells and prevent poisoning [13]. Proteins have natural fluorescence ability because of their aromatic amino acid residues, especially tryptophan and tyrosine[14]. The indole nucleus of the tryptophan is very sensitive fluorofore. Thus, fluorescence spectroscopy has been proven to be a widely useful technique for the characterization of protein-ligand interaction [15,16]. However, while numerous studies have been reported about different parameters related to different ligands interaction to serum albumin [17,18], so far no studies reported about the different binding and theremodynamical parameters of Oval ligands interaction. Therefore, this preliminary study aims to investigate OA conjugation to Oval by spectrofluorimetric method at different temperature values. MATERIALS and METHODS All chemicals used in this study were of analytical grade, purchased from a variety of sources and used without further purification. Oval was from Sigma (A-5253), and Oleic acid was a gift from Biology dept., IUG. Preparation of Oval/OA conjugate Oval (6.5 µm) was prepared in 10 mm phosphate buffer solution, ph 6.9. Oval concentration was measured spectrophotometrically using the absorption coefficient of M -1 Cm -1 at 280 nm [19]. OA (3 mm) was prepared in the same buffer containing 5 % ethanol in order to obtain complete solubility. Conjugation was carried out by successive addition of 10 µl of the OA solution to 2 ml of the Oval solution at each temperature. Fluorescence measurements Fluorescence measurements of the conjugate were carried out by using a Perkin Elmer luminescence (series no ) spectrometer equipped with a water jacketed cuvette holder

3 that was maintained at constant temperature by means of a circulatory water bath. The temperatures range used was C. The conjugation of OA with Oval was monitored by measuring the intrinsic fluorescence at maximum emission wavelength as a function of OA concentration. However, each of the conjugate preparation was excited at 370 nm and the maximum emission spectra after 3 min of incubation were recorded in the wavelength range of nm. Slits of 10 nm for both excitation and emission were used. All conjugation data reported throughout the study corresponded to the average of two values. Data analysis Estimation of binding and thermodynamic parameters from the obtained data were performed using Origin Software (Version 5) RESULTS AND DISCUSSION Fluorescence intensity of Oval/OA conjugates Figure 1 shows that the observed fluorescence spectra of the conjugate at 33 C displayed a broad band centered at 445 nm. It can be clearly seen that the fluorescence emission maxima at the mentioned wavelength increase as the [OA]/[Oval] ratio increases. Hence, successive additions of OA aliquots induce an increase in the fluorescence intensity without a shift in the maximum emission wavelength. Figure 2 shows a representative binding curve, depicting the change in fluorescence intensity (FI) accompanying the conjugation of OA as a function of the total OA concentration at 22 0 C. Similar binding hyperbolic curves were also obtained at other temperatures. Calculation of binding parameters: The K a and the binding capacity of the Oval/OA conjugate were estimated from the Scatchard equation [20] using The following simple equilibrium: OA + Oval OA-Oval [ OA Oval] Where K a = (1) [ OA][ n ( Oval)] [OA], [OA-Oval] and n represent concentration of free OA, concentration of bound OA and number of binding sites of OA in the Ovalbumin, respectively. Rearrangement of the equation (1) yields [ OA Oval] [ OA][ Oval] T = n K a K a [ OA Oval] [ Oval] T (2) Where, [Oval] T represents the total ovalbumin concentration. After introducing the coupling parameter r = [OA-Oval]/[Oval] T, equation (2) can be rearranged to get the following Scatchard equation : r = n K a K ar (3) [ OA] free A plot of r/[oa] free against r gave the Scatchard plot whose slope is K a and X-intercept equals the number of binding sites on the protein [Figure 3). Linear Scatchard plots were also constructed at other temperatures which indicated the presence of independent binding sites of OA in the ovalbumin [21] throughout the range. Table 1 shows that average n and K a values at 22 C were about 2.0 and M 1, respectively. At 55 C, n and K a values

4 were about 3.0 and 0.73 x 10 3 M 1, respectively. In contrast to the binding capacity, association constants become lower as the temperatures increase.

5 Calculation of thermodynamic parameters. The association constant of the conjugate was used for calculation of the corresponding Gº value at each temperature. Moreover, the temperature dependence of the k a for the conjugate was analyzed by Van't Hoff plot of LnK a vs 1/T and linear fit was obtained. From the slope of the straight line, the value of Van't Hoff enthalpy ( H VH ) was calculated. The entropy change ( Sº) was calculated from the X-intercept of the line. The results showed that, the interaction was enthalpically driven with a negative value of Sº. The negative value of H VH can be explained by the formation of H-bonding and Van der Waals forces during the conjugation [16,22]. The unfavorable entropic contribution may arise from the loss of vibration degrees of freedom of oval domains comprising the OA ligands. Thus, for stable binding to occur, the enthalpic contribution must be sufficient high to offset of unfavorable entropy of the conjugate [23]. Conclusion In a conclusion, the total OA capacity of Oval and the resultant K a of the conjugate varied with temperature values. The average OA binding sites in Oval were ranged from two to three per Oval molecule. Such conjugation of OA to Oval was independent, enthalpically driven and associated with negative value of the entropy. REFERENCES [1] Huntington, J.A., and Stein, P.E. Structure and properties of Oval. J. Chromatography B, 756, (2001). [2] Hunt, L.T., and Dayhoff, M.O. A surprising new protein superfamily containing Ova, antithrombin-iii, and Alpha 1-proteinase inhibitor. Biochem. Biophys. Res. Commun., 95(2), (1980). [3] Nisbet, A.D., et al. The complete amino acid sequence of hen ovalbumin. Eur. J. Biochem., 115, , (1981). [4] McReynolds, L., et al. Sequence of chicken ovalbumin mrna. Nature, 273, (1978). [5] Tai, T., et al. Structures of the carbohydrate moiety of ovalbumin glycopeptide III and the difference in specificity of end-beta-n-acetylgluosaminidases CII and H. J. Biol. Chem., 252, (1977). [6] Donovan, J. W. and Mapes, C. K. A differential scanning calorimetric study of conversion of ovalbumin to s-ovalbumin in eggs. J. Sci. food Agric., 27, (1976). [7] Harlow, E. and Lane, D. Antibodies: A lab Manual. Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY) 77 (1988). [8] Peters, T., Jr., and Davidson, L.K. The biosynthesis of rat serum albumin. In vivo studies on the formation of the disulfide bonds. J. Biol. Chem., 257, (1982). [9] Fuller Noel, J.K. and Hunter, M.J. Bovine mercabtalbumin and non-mercaptalbumin monomers. Interconversions and structural differences. J. Biol. Chem., 247, (1972). [10] Oliveira, F.M., et al. Covalent coupling of palmitate to Ovalbumin inhibits and blocks the induction of oral tolerance. Scand. J. Immunol. 55, (2002). [11] Mowat A., M., Depletion of suppressor T cell by 2-deoxyguanosine abrogates tolerance in mice fed Oval and permits the induction of intestinal delayed-type hypersensitivity. Immunology, 49, (1983). [12] Oliveira, F.M., et al. Coupling of palmitate to Ovalbumin inhibits the induction of oral tolerance. Barz. J Med. Biol. Res., 31, (1998). [13] Baynes, J. W., Dominiczak, M.H., Medical biochemistry, 2nd edition, 59 (2005).

6 [14] Chen, R. F. Fluorescence: Theory, instrumentation, and practice, Dekker, New York. (1967). [15] Birdsall, B., et al., Correction for light absorption in fluorescence studies of proteinligand interactions. Anal. Biochem., 132, (1983). [16] Connelly, P.R., et al. Probing hydration contributions to the thermodynamics of ligand binding by proteins. Biochem., 32, (1993). [17] Peyrin, E.; Guillaume, Y.C., and Guinchard, C. Characterization of solute binding at human serum albumin sit II and its geometry using biochromatographic approach. Biophys. J., 77(3) (1999). [18] Ferrer, M.L., et al. The Conformation of serum albumin in solution: Modeling study. Biophys. J., 80 (5) (2001). [19] Fasman, G.D. Practical Handbook of Biochemistry and Molecular Biology, CRC Press (Boca Raton, FL) (1989). [20] Weder, H.G., et al. Determination of binding parameters from Scatchard plots: theoretical and practical considerations. Eur. J. Biochem. 42, (1974). [21] Chen Y., et. al. Probing ligand protein binding equilibria with fluorescence fluctuation spectroscopy. Biophys J., 79 (2), (2000). [22] Connelly,P.R., et al. Thermodynamics of protein-peptide interactions in the ribonuclease S. system studied by titration Calorimetry. Biochem., 29(25), (1990). [23] Eftink, M.R., et al. Enthalpy-entropy compensation and heat capacity changes for protein-ligand interactions: general thermodynamical models and data for the binding of nucleosides to ribonuclease A. Biochem., 22, (1983).

7 Fluorescence Intensity Arbitrary Units Wavelength, nm Figure1: Four representative fluorescence emission spectra of Oval/OA conjugate in phosphate buffer solution ph 6.9 at 33 C. The lines in the direction of arrow correspond to successive additions of OA aliquots FI (arbitrary units) [OA] total, mm Figure 2: Binding curve of Oval/OA conjugate in the phosphate buffer at 22 C (λ exc = 370 nm, and λ em = 445 nm). Change in FI was plotted as a function of total OA concentration.

8 Figure 3: Scatchard plot of Oval/OA conjugate at 22 C r/[oa] free x r

9 Table1: Binding and thermodynamic parameters of Oval/OA conjugate at different temperatures. T (K) K a 10 3 (M -1) n Gº ( kj/mol ) H VH (kj/mol) T Sº (kj/mol) -6.58