COMPLEXATION OF BOVINE SERUM ALBUMIN WITH CATIONIC POLYELECTROLYTES AT ph 7.40. FORMATION OF SOLUBLE COMPLEXES. Theofanis Asimakopoulos, Georgios Staikos Department of Chemical Engineering, Universty of Patras, GR 26504, Patras, Greece ABSTRACT Complexation of Bovine Serum Albumin, (BSA), with polycations was investigated at ph = 7.40, in water, and in 0.15 M NaCl, as a function of the polycation/bsa charge ratio, r. Turbidity, UV absorption, potentiometry, ζ- potential and static scattering were used to indicate the formation of insoluble stoichiometric complexes due to Coulombic interactions. INTRODUCTION Interactions between proteins and polyelectrolytes have attracted a considerable interest 1-3. The most critical parameter is the solution ph, relatively to the protein isoelectric point (IP). Coulombic interactions between a protein and a polyelectrolyte lead to phase separation through the formation of complexes at ph lower than IP with polyanions and higher than IP with polycations 4-8. Many studies have been focused in a wide range of ph and in various ionic strengths 9-11, examining the complex coacervation 7, 9, 12, 13, as well as the size and mobility of the complexes formed 4, 11, 14. Moreover such a complex formation while quite noticeable at low ionic strength, is barely observable at high 8, 11. Even if phase separation and complex coacervation is a usual behavior, soluble complexes are formed with bovine serum albumin, (BSA), through Coulombic interactions instead of a complex coacervation 15, 16. In this study, we aim to investigate complexation of BSA with a cationic polyelectrolyte, poly([3-(methacryloylamino)propyl]trimethylammonium chloride) (PMAPTAC) and with a graft copolymer of PMAPTAC with poly(n, N-dimethylacrylamide) (PDMAM), PMAPTAC-g-PDMAM75, at ph 7.40 in water and in 0.15 M NaCl, close to the physiological conditions, as a function of the polycation/protein charge ratio, r. EXPERIMENTAL METHODS Bovine serum albumin (BSA) was purchased from Sigma (A-7638) and used without further purification. The monomers (MAPTAC) and (DMAM), were purchased from Aldrich. Ammonium persulfate (APS, Aldrich), potassium metabisulfite (PBS, Aldrich), 2-aminoethanethiol hydrochloride (AET, Aldrich) and 1-(3- (dimethylamino)propyl)-3-ethyl-carbodiimide hydrochloride (EDC, Aldrich) were used for the synthesis of the polymers. The polymers used were synthesized by free radical polymerization at 30 o C using a redox couple and purged with N 2 for 30 minutes. For the turbidity, absorption and potentiometry measurements, mother solutions of BSA 0.1 mm and polycations 50 mm were used. These molar concentrations were based on the molecular mass per charge unit. It was taken equal to 3690 Da per charge for BSA (M = 66400 Da 17, 18 negative charges at ph 7.40 18 ), 220.5 for PMAPTAC and 890 for PMAPTAC-g-PDMAM75. The BSA/polycation mixtures in solution were prepared by adding the appropriate volume of the cationic polyelectrolyte solution in 10 cm 3 of a 0.1 mm BSA solution. The buffer solution used, ph = 7.40, was prepared by mixing 0.02 M Tris solution in water or in 0.15 M NaCl, with 0.02 M HCl in a 100 to 76 v/v ratio.
RESULTS AND DISCUSSION BSA/PMAPTAC interactions at ph = 7.40 in water and in 0.15 M NaCl. From the results obtained the turbidity (optical density), Figure 1, increases as r increases, reaching a maximum at r 1.5 and then, after decreasing a little, it reaches a new maximum at r 3, decreasing again at low optical density values for higher r. The turbidity appeared indicates a phase separation as a result of the formation of insoluble BSA/PMAPTAC complexes, due to Coulombic interactions. Even if a turbidity maximum at r = 1 should be expected, the shift of this maximum at a broader charge ratio range, r = 1.5 3 should be attributed to a further dissociation of the carboxylic acid groups of the protein, due to its binding with the polycation and the formation of a protein/polyelectrolyte complex, accompanied with proton release and a decrease of the pk a of the protein carboxylic groups. This assumption is supported by zeta potential measurements which show that the stoichiometry is close to r 2 instead of 1. On the contrary to what happens in water, in 0.15 M NaCl, corresponding to the ionic strength at physiological conditions, the BSA/PMAPTAC solution mixtures remain clear, i.e. Optical Density = 0, for all the range of the polycation/bsa charge ratio studied. This behavior shows that in 0.15 M NaCl, either there is not any complexation between BSA and PMAPTAC, due to the charge shielding effect, or water soluble BSA/PMAPTAC complexes are formed. Optical Density (a.u.) 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0-0.2 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Figure 1. Variation of the turbidity of BSA/PMAPTAC mixtures at ph = 7.40, in water ( ), and in 0.15 M NaCl ( ), versus the. In order to further elucidate the complex formation between BSA and PMAPTAC at ph = 7.40, in water and in 0.15 M NaCl we have measured the absorbance of the solutions at 280 nm after centrifugation at 5000 rpm so that clear solutions in both cases were taken. The results obtained, are shown in Figure 2(a) where the BSA fraction, remaining in solution after precipitation of any insoluble BSA/PMAPTAC complex formed, is shown as it has been measured through the UV absorbance ratio relatively to the value of the pure BSA solution in water or NaCl 0.1M. We observe that in water, BSA fraction decreases rapidly, showing a minimum, where most of the BSA should have been phase separated as an insoluble complex with PMAPTAC, while as r increases, BSA fraction increases considerably, showing that formation of soluble non stoichiometric BSA/PMAPTAC complexes prevails. In 0.15 M NaCl the BSA fraction remains stable, as in the pure BSA solution, indicating that at physiological conditions, an insoluble complex between BSA and PMAPTAC is not formed. Nevertheless a question remains, if in these conditions soluble BSA/PMAPTAC complexes are formed. In order to answer to the above question, we carried out a potentiometric study as follows. In a 10 ml, 0.1 mm BSA solution in pure water, after adjusting its ph with 0.01 M NaOH at ph equal to 7.40, a 50 mm PMAPTAC solution in water was gradually added with a micropipette. According to the results obtained shown in Figure 2 (b), a substantial decrease in ph was observed versus the polycation/bsa charge ratio, r, from ph = 7.40 at r = 0, to ph = 6.22, at r = 5. These results are explained by proton release from BSA, as a protein/polycation complex is formed, due to Coulombic interactions, while the solution becomes turbid due to the formation of an insoluble BSA/PMAPTAC complex.
BSA fraction 1,2 1,1 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 PMAPTAC/BSA charge ratio, r ph 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 PMAPTAC/BSA charge ratio, r Figure 2: (a) Variation of the BSA fraction of BSA/PMAPTAC solution mixtures at ph = 7.40, in water ( ), and in 0.15 M NaCl ( ) after centrifugation, versus the.(b) Variation of ph of BSA/PMAPTAC mixtures in solution in water ( ), and in 0.15 M NaCl ( ), versus the Polycation/BSA charge ratio, r. On the contrary, in 0.15 M NaCl, ph was stable, while the solution remained clear in all the charge ratio range studied, showing that not any protein/polycation complex, soluble or not, was formed. BSA/PMAPTAC-g-PDMAM75 interactions at ph = 7.40 in water and in 0.15 M NaCl In order to further investigate the protein/polycation complex formation, we carried out a potentiometric study of the mixtures of BSA with a copolymer of PMAPTAC grafted with neutral hydrophilic PDMAM side chains, PMAPTAC-g-PDMAM75, in water and in 0.15 M NaCl, starting with a BSA solution at ph = 7.40, given that all these mixtures formed clear solutions in all the charge ratio range studied, r = 0 5. The potentiometric results obtained are shown in Figure 3(a). In water, ph decreases considerably from 7.40 at r = 0, to 6.57 at r = 5, indicating the formation of a soluble complex between BSA and PMAPTAC-g-PDMAM75. Moreover, we observe that the ph decrease in the case of the graft copolymer, Figure 3 (a), is slightly lower than that of the homopolymer, Figure 2 (b). ph reaches a higher value at r = 5, equal to 6.57, as it compares with a value equal to 6.22, in the case of the homopolymer. This behavior should be due to a weaker complexation in the case of the graft copolymer due to a decrease of the charge density of the polycation chain. In 0.15 M NaCl, ph remains stable in all the charge ratio range studied indicating that not any complex is formed. This behavior should be attributed to the charge shielding effect, as a result of the ionic strength of the solution. Figure 3 (b) shows the ζ-potential variation of the BSA/PMAPTAC-g-PDMAM75 mixtures in ph =7.40 in water and in 0.15 M NaCl as a function of the PMAPTAC-g-PDMAM75/BSA charge ratio, r. We see that in water the ζ-potential values obtained varies from negative values at low r characteristic of the negative charge of BSA at ph = 7.40 to positive values at r > 2.75 indicating the formation of a BSA/PMAPTAC-g-PDMAM75 complex formation positively charged due to an excess of the cationic polyelectrolyte. The value of r = 2.75 where ζ-potential becomes zero should correspond to a stoichiometric complex i.e. in which BSA carboxylate groups have increased from 18 to 50 explaining the proton release already observed in the potentiometric study. However in 0.15 M NaCl the ζ-potential remains negative in all the BSA/PMAPTAC-g-PDMAM75 compositions showing that at this ionic strength the negatively charged BSA molecules remain uncomplexed.
ph 7,5 7,4 7,3 7,2 7,1 7,0 6,9 6,8 6,7 6,6 6,5 6,4 6,3 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 Zeta Potential, mv 10,0 7,5 5,0 2,5 0,0-2,5-5,0-7,5-10,0-12,5-15,0-17,5-20,0 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 Figure 3: (a) ph variation of BSA/PMAPTAC-g-PDMAM75 mixtures in solution in water ( ) and in 0.15 M NaCl ( ) versus the Polycation/BSA charge ratio r. (b) ζ potential of BSA/PMAPTAC-g-PDMAM75 mixtures in solution in water ( ) and in 0.15 M NaCl ( ) versus the Polycation/BSA charge ratio r. In order to confirm the formation of the soluble complexes between BSA and the graft copolymer we carried out a static light scattering study. Figure 4 shows the intensity ratio ΔΙ exp/δi id as a function of the polycation/bsa charge ratio, r, at ph=7.40 in water and in NaCl 0.15M, where ΔI exp is the difference of the light intensity scattered at 90 o between the mixture solution and the solvent, and ΔI id is the ideal value, calculated additively by the intensity difference values of the BSA and the PMAPTAC-g-PDMAM75 solutions. 3.5 3.0 ΔI exp /ΔI id 2.5 2.0 1.5 1.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Figure 4: ΔI exp/δi id variation of BSA/PMAPTAC-g-PDMAM75 mixtures in solution in water ( ), and in 0.15 M NaCl ( ) versus the. From the results obtained, in water, intensity ratio, ΔI exp/δi id increases rapidly at about 3.0 for r =1.5 and then reaches a plateau. This behavior is indicative of core shell colloidal particles formation between BSA and PMAPTAC-g-PDMAM75, of a certain stoichiometry. Again zeta potential measurements place stoichiometry close to r 2.75. On the contrary, in NaCl 0.15M the light intensity ratio remains practically stable, close to 1 for all the charge ratio range examined, showing an ideal behavior indicative of the absence of any BSA graft copolymer complexation in accordance with the potentiometric results, confirming again that the charge shielding effect, at this physiological ionic strength value, is strong enough to prevent the protein-polycation Coulombic interaction.
CONCLUSIONS In this work, we have investigated the interactions of BSA with a cationic homopolymer, PMAPTAC, and a graft copolymer, PMAPTAC-g-PDMAM75, in order to study the formation of BSA/Polycation complexes at ph = 7.40 in water, and in 0.15 M NaCl, close to physiological conditions. Turbidimetric and UV absorption measurements have shown that in the case of the homopolymer in water a phase separation occurs, due to the formation of insoluble BSA/PMAPTAC complexes, as a result of Coulombic interactions in accordance with literature 4, 8, 19, 20. Potentiometric measurements corroborate this conclusion, showing proton release as a result of Coulombic interactions of the polycation with the BSA carboxylate groups. On the other hand, at 0.15 M NaCl, according to the turbidity and UV absorbance results, insoluble complexes are not formed, but the formation of soluble complexes should not be excluded, in principle. However, potentiometric measurements exclude the formation of such complexes in 0.15 M NaCl, that it has not been clearly stated in literature, while their formation in water is further confirmed. In the case of BSA mixtures with the graft copolymer, where only clear solutions are formed, both in water and in 0.15 M NaCl, potentiometric and static light scattering measurements show that BSA/PMAPTAC-g- PDMAM75 soluble complexes are formed only in water, while ζ-potential is further corroborating this conclusion, providing an indication of their stoichiometry. Molecular weight determination, by static light scattering measurements, confirms the formation of the above soluble complexes in water in a stoichiometry practically the same with that indicated by ζ-potential results.
REFERENCES [1]. Cooper C.L., Dubin P.L., Kayitmazer A.B., Turksen S. Curr. Opin. Colloid Interface Sci. (2005), 10, p.52-78. [2]. Becker A.L., Henzler K., Welsch N., Ballauf M., Borisov O. Curr. Opin. Colloid Interface Sci. (2012), 17, p.90-96. [3]. Kayitmazer A.B., Seeman D., Baykal Minsky B., Dubin P.L., Xu Y. Soft Matter. (2013), 9, p.2553-2583. [4]. Mattison K.W., Wang Y., Grymonpre K., Dubin P.L. Macromol. Symp. (1999), 140, p.53-76. [5]. Li Y., Mattison K.W., Dubin P.L., Havel H.A., Edwards S.L. Biopolymers. (1996), 38, p.527-533. [6]. Mattison K.W., Dubin P.L., Brittain I.J. J. Phys. Chem. (1998), B, 102, p.3830-3836. [7]. Kaibara K., Okazaki T., Bohidar H.B., Dubin P.L. Biomacromolecules. (2000), 1, p.100-107. [8]. Wen Y.P., Dubin P.L. Macromolecules. (1997), 30, p.7856-7861. [9]. Ahmed L.S., Xia J., Dubin P.L., Kokufuta E. J. M. S. Pure Appl. Chem.(1994), A31(1), p.17-29. [10]. Matsunami H., Kikuchi R., Ogawa K., Kokufuta E. Colloids Surf. B. (2007), 56, p.142-148. [11]. Ball V., Winterhalter M., Schwinte P., Lavalle Ph., Voegel J.C., Schaaf P. J. Phys. Chem. B. (2002), 106, p.2357-2364. [12]. Wang Y., Gao J.Y., Dubin P.L. Biotechnol. Prog. (1996), 12, p.356-362 [13]. Xu Y., Mazzawi M., Chen K., Sun L., Dubin P.L. Biomacromolecules. (2011), 12, p.1512-1522. [14]. Xia J., Dubin P.L., Dautzenberg H. Langmuir. (1993), 9, p.2015-2019. [15]. Sotiropoulou M., Bokias G., Staikos G.. Biomacromolecules. (2005), 6, p. 1835-1838. [16]. Serefoglou E., Oberdisse J., Staikos G.. Biomacromolecules. (2007), 8, p.1195-1199. [17]. Hirayama K., Akashi S., Furuya M., Fukuhara K. Biochem. Biophys. Res. Commun. (1990), 173, p.639-646. [18]. Tanford C. J. Am. Chem. Soc. (1950), 72, p441 451. [19]. Klemmer K.J., Waldner L., Stone A., Low N.H., Nickerson M.T. Food Chem. (2012), 130, p.710-715. [20]. Wang Y-fan., Gao J.Y., Dubin P.L. Biotechnol. Prog. (1996), 12, p.356-362.