Interaction of piroxicam with bovine serum albumin investigated by spectroscopic, calorimetric and computational molecular methods.

The binding of drugs to serum proteins is governed by weak non-covalent forces. In this study, the nature and magnitude of the interactions between piroxicam (PRX) and bovine serum albumin (BSA) was assessed using spectroscopic, calorimetric and computational molecular methods. The fluorescence data revealed an atypical behavior during PRX and BSA interaction. The quenching process of tryptophan (Trp) by PRX is a dual one (approximately equal static and dynamic quenched components). The FRET results indicate that a non-radiative transfer of energy occurred. The association constant and the number of binding sites indicate moderate PRX and BSA binding. The competitive binding study indicates that PRX is bound to site I from the hydrophobic pocket of subdomain IIA of BSA. The synchronous spectra showed that the microenvironment around the BSA fluorophores and protein conformation do not change considerably. The Trp lifetimes revealed that PRX mainly quenches the fluorescence of Trp-213 situated in the hydrophobic domain. The CD and DSC investigation show that addition of PRX stabilizes the protein structure. ITC results revealed that BSA-PRX binding involves a combination of electrostatic, hydrophobic and hydrogen interactions. The analysis of the computational data is consistent with the experimental results. This thorough investigation of the PRX-BSA binding may provide support for other studies concerning moderate affinity drugs with serum protein.Communicated by Ramaswamy H. Sarma.

Adsorption of branched-linear polyethyleneimine-ethylene oxide conjugate on hydrophilic silica investigated by ellipsometry and Monte Carlo simulations.

The adsorption of and conformation adopted by a branched-linear polymer conjugate to the hydrophilic silica-aqueous solution interface have been studied by in situ null ellipsometry and Monte Carlo simulations. The conjugate is a highly branched polyethyleneimine structure with ethyleneoxide chains grafted to its primary and secondary amino groups. In situ null ellipsometry demonstrated that the polymer conjugate adsorbs to the silica surface from water and aqueous solution of 1 mM asymmetric divalent salt (calcium and magnesium chloride to emulate hard water) over a large pH range. The adsorbed amount is hardly affected by pH and large charge reversal on the negatively charged silica surface occurred at pH = 4.0, due to the adsorption of the cationic polyelectrolyte. The Monte Carlo simulations using an appropriate coarse-grained model of the polymer in solution predicted a core-shell structure with no sharp boundary between the ethyleneimine and ethyleneoxide moieties. The structure at the interface is similar to that in solution when the polymer degree of protonation is low or moderate while at high degree of protonation the strong electrostatic attraction between the ethyleneimine core and oppositely charged silica surface distorts the ethyleneoxide shell so that an "anemone"-like configuration is adopted. The adsorption of alkyl benzene sulfonic acid (LAS) to a preadsorbed polymer layer was also investigated by null ellipsometry. The adsorption data brought additional support for the existence of a strong polymer adsorption and showed the presence of a binding which was further enhanced by the decreased solvency of the surfactant in the salt solution and confirmed the surface charge reversal by the polymer adsorption at pH = 4.0.

Monte Carlo simulations of flexible polyelectrolytes inside viral capsids with dodecahedral charge distribution.

Structural properties of encapsidated flexible polyelectrolytes in viral capsids with dodecahedral charge distribution have been investigated by Monte Carlo simulations using a coarse-grained model. Several capsid charge distributions ranging from a homogeneous surface charge distribution (lambda=0) to a complete dodecahedral distribution (lambda=1) at constant total capsid charge and fixed radial location of the capsid charges have been considered. The radial and lateral organizations of the polyelectrolyte have been examined as a function of the polyelectrolyte length and capsid charge distribution. With short polyelectrolytes a single polyelectrolyte layer was formed at the inner capsid surface, whereas at increasing polyelectrolyte length also a uniform polyelectrolyte density inside the surface layer was established. At low lambda , the polyelectrolyte layer was laterally isotropic, but at lambda> or =0.05 a dodecahedral structure started to appear. At lambda=1 , the polyelectrolyte followed essentially a path along the edges of a dodecahedron. With sufficiently long chains, all edges were decorated with polyelectrolyte, facilitated by loop formation. For an undercharged capsid, the capsid counterions inside the capsid also adopted a dodecahedral distribution.

Packaging of a flexible polyelectrolyte inside a viral capsid: effect of salt concentration and salt valence.

The effect of salt on the location and structure of a flexible polyelectrolyte confined inside a viral capsid and the Donnan equilibrium of the salt across the capsid have been examined using a coarse-grained model solved by Monte Carlo simulations. The polyelectrolyte was represented by a linear jointed chain of charged beads, and the capsid was represented by a spherical shell with embedded charges. At low salt concentration, the polyelectrolyte was strongly adsorbed onto the inner capsid surface, whereas at high salt concentration it was located preferentially in the central part of the capsid. Under the condition of equal Debye screening length, the electrostatic screening increased as the valence of the polyelectrolyte counterion was increased. The distribution of the small cations and anions was unequal across the capsid. An excess of polyelectrolyte counterions occurred inside the capsid, and the excess increased with the salt concentration. A simplified representation of the small ions through the use of the screened Coulomb potential provided only a qualitatively correct picture; the electrostatic screening originating from the small ions was exaggerated.

Monte Carlo simulations of polyelectrolytes inside viral capsids.

Structural features of polyelectrolytes as single-stranded RNA or double-stranded DNA confined inside viral capsids and the thermodynamics of the encapsidation of the polyelectrolyte into the viral capsid have been examined for various polyelectrolyte lengths by using a coarse-grained model solved by Monte Carlo simulations. The capsid was modeled as a spherical shell with embedded charges and the genome as a linear jointed chain of oppositely charged beads, and their sizes corresponded to those of a scaled-down T=3 virus. Counterions were explicitly included, but no salt was added. The encapisdated chain was found to be predominantly located at the inner capsid surface, in a disordered manner for flexible chains and in a spool-like structure for stiff chains. The distribution of the small ions was strongly dependent on the polyelectrolyte-capsid charge ratio. The encapsidation enthalpy was negative and its magnitude decreased with increasing polyelectrolyte length, whereas the encapsidation entropy displayed a maximum when the capsid and polyelectrolyte had equal absolute charge. The encapsidation process remained thermodynamically favorable for genome charges ca. 3.5 times the capsid charge. The chain stiffness had only a relatively weak effect on the thermodynamics of the encapsidation.