Acta Biophysica Romana 2006 22-24 Febbraio Università di Roma - Tor Vergata Protein Synthetic Lipid Interactions Silvia Tardioli and Adalberto Bonincontro CNISM-Dipartimento di Fisica Camillo La Mesa Dipartimento di Chimica Research Center SOFT-INFM-CNR Università La Sapienza
Table of contents Bovine Serum Albumin (BSA) Gemini Surfactants: C6A2 and C2A2 BSA-Gemini Surfactants Interactions: experimental procedures ph=7.2: BSA-C6A2 and BSA-C2A2 systems ph=4.4: BSA-C6A2 and BSA-C2A2 systems Results Conclusions
Bovine Serum Albumin (BSA) Globular protein, carrier of fatty acid in the blood. Not a specific biological activity. Single polypeptide chain of 585 amino-acid (homology with HSA:70%) arranged in a multiple loop structure: α - helix 68 %. Rigid oblate ellipsoid: minor semi-axes b=c= 2 nm; a=3.5 nm. The 3 domains of BSA including the calculated net negative charge at ph=7.4 equivalent to 19 electrons. Isoelectric point : ph=4.8.
Gemini Surfactants New type of dimeric surfactant. Two monomeric surfactant molecules linked by a spacer chain. S can vary in length, hydrophobicity and flexibility. They may be considered the synthetic homologue of phospholipids Two cationic compounds with the same monomeric units but different spacer lenght: C6A2 (S=6 methile groups) C2A2 (S=2 methile groups)
BSA-Gemini Surfactants Interactions Not much is known yet on the interaction processes between Gemini surfactants and proteins. We present a study of the mechanisms of binding of two ionic Gemini surfactants with BSA. Objectives Role of the driving forces in the binding. Defining the saturation level of Gemini surfactants per BSA molecule. Evaluation of the conditions of the development of protein aggregates.
Experimental procedures ph 7.2 4.4 7.2 (BSA Charge: -19e) Aqueous solutions at two ph values 4.4 (BSA Charge: +11e) BSA concentration 0.27mM; measurements at different SURFACTANT/BSA molar ratios: from one dimer per protein up to a charge ratio 1:1. Complementary techniques have been employed: DS, DLS, CD, UV, Zeta potential.
BSA-C6A2 and BSA-C2A2 interactions ph=7.2: evaluation of the effect of the spacer lenght in the mechanisms of interaction. ph=4.4: hydrophobic interactions.
ph=7.2 : BSA-C6A2 and BSA-C2A2 systems ph 7.2 In literature changes in the secondary structure of BSA caused CD spectra by binding of ionic surfactants have been reported. [θ] 10-5(deg cm 2 decimol-1) 40 BSA-C6A2 0 Our 0 CD measurements show a variation in the α-helix content of BSA. -40-80 200 210 220 230 240 Specific Binding starts at 1 dimer surfactant per BSA molecule. λ nm [θ] 10-5 (deg cm2decimol -1 ) 40-40 -80 200 210 220 230 240 λ nm BSA-C2A2
ph=7.2 : BSA-C6A2 and BSA-C2A2 systems ph 7.2 Turbidity at low SURFACTANT/BSA molar ratios. Variation in the dielectric properties of the systems studied as a function of the Gemini surfactant concentration is useful to evaluate a critical molar ratio R, for aggregation phenomena.
16 12 BSA-C6A2 ph=7.2 : dielectric relaxation data ε '' d 8 4 16 BSA-C2A2 12 ε d '' 8 4 ph 7.2 ε' ε 8 4 0 10 5 10 6 10 7 10 8 ν Hz ε' ε 8 4 0 10 5 10 6 10 7 10 8 ν HZ 0 10 6 10 7 10 8 ν Hz Dielectric permittivity ε, normalized to ε, for BSA-Surfactant solutions at different molar ratios. In the inset relaxation dielectric loss. 10 6 10 7 10 8 ν HZ Solid lines:cole-cole equation fitted to the data. Fit parameters : ε (dielectric increment) and ν (relaxation frequency). 0
ph=7.2 : ε as a function of C6A2 concentration ph 7.2 ε 16 12 8 4 0 R=7 0 2 4 6 8 10 [C6A2]/[BSA] Up to 7: Dielectric relaxation of BSA-C6A2 complexes can be ascribed to orientational polarization. Above 7: Dielectric relaxation of aggregates can be ascribed to motion of counter-ions at interface.
ph=7.2 : ε as function of C2A2 concentration ph 7.2 ε 16 12 8 4 Up to 4: Dielectric relaxation of BSA-C2A2 complexes can be ascribed to orientational polarization. R=4 Above 4: Dielectric relaxation of aggregates can be ascribed to motion of counter-ions at interface. 0 0 2 4 6 8 10 [C2A2]/[BSA]
6 ν as a function of C6A2 and C2A2 concentration 6 ph 7.2 ν MHz 5 4 3 ν MHz 5 4 3 2 0 2 4 6 8 10 [C6A2]/[BSA] 0 2 4 6 8 10 [C2A2]/[BSA] According to Debye theory: up to R=7 and R=4 no variation in BSA hydrodynamic radius is evidenced. Dielectric response of the aggregates is indicative of 5-10 nm in terms of equivalent sphere (Barchini-Pottel model). 2
10 ph=7.2: Dynamic Light Scattering Data Plateau at 9 nm: aggregates composed of BSA dimers in solution. ph 7.2 < 2R H > nm 8 6 4 2 0 2 4 6 8 10 [SURFACTANT]/[BSA] DLS results in agreement with R from DS: 7 (BSA-C6A2) and 4 (BSA-C2A2). BSA-C6A2 BSA-C2A2 BSA BSA-C6A2 complexes are smaller than BSA-C2A2
< Ζ > mv -8-12 ph=7.2: Zeta Potential Data Z potential is a controlling parameter in BSA-Surfactant binding. BSA-C 6 A 2 BSA-C 2 A 2 BSA-C6A2: charge neutrality of BSA-BSA aggregates. ph 7.2 Measurements indicate the variation of the alteration on protein charge by adding Gemini surfactants. Z value decreases: counterions progressively released 0 by the binding of Gemini on to the BSA surface. -4 2 4 6 8 10 [SURFACTANT]/[BSA] C6A2 and C2A2 are equal in monomeric units. The different behaviour is due to the different spacer lenght.
ph=4.4: BSA-C6A2 and BSA-C2A2 systems CD Data: reversible variation in the helical content due to the binding of C6A2 and C2A2 starting from 1 dimer per BSA Formation of complexes in which tails of Gemini surfactants bind in the hydrophobic patches of BSA ph 4.4 < Ζ > mv 25 20 15 10 BSA-HCl-C6A2 BSA-HCl-C2A2 BSA-HCl 0 2 4 6 8 [SURFACTANTS]/[BSA] Zeta Potential Data: Z value increases with the number of dimeric surfactant per protein molecules. The counter-ion distribution progressively increases with the number of dimeric surfactant bind to BSA.
< 2R H > nm 8 7 6 5 4 ph=4.4: Dynamic Light Scattering Data BSA-HCl-C6A2 BSA-HCl-C6A2 2 4 6 8 [SURFACTANT]/[BSA] ph 4.4 Mean dimension for both systems under 8 nm. No BSA dimers formation. BSA-C6A2 complexes are smaller than BSA-C2A2 as at ph=7.2. C6A2 and C2A2 are equal in monomeric units. The differences in size of the complexes are due to the different spacer lenght.
Results 1/2 ph=7.2: BSA strongly interacts with oppositely charged surfactants in water by electrostatic attraction between the head groups of the surfactants and the charge protein binding sites. BSA has hydrophobic patches (4). Hydrophobic binding is not excluded but at a minor extent. As the surfactant concentration increases aggregation occurs long before the total charge saturation of BSA: BSA-BSA aggregation is mediated by the tails of the Gemini surfactants adsorbed on two different proteins. ph=4.4: both Gemini surfactants and BSA are positively charged: data from different tecniques all agree in the formation of complexes in which tails of Gemini surfactants bind in the hydrophobic patches of BSA.
Results 2/2 The spacer lenght controls the critical molar ratio R, and the mean size of BSA-Gemini surfactant complexes. From CD Spectra: Gemini surfactants are high denaturant agents.
Conclusions Bio-medical field: novel Gemini surfactants as synthetic vectors for the safe delivery of functional genes into cells and then into the nucleus. However our measurements show critical problems in the use of these molecules: Gemini surfactants influence the secondary strucutre of BSA. Gemini surfactants induce protein-protein aggregation.