Conformational flexibility of lipase Lip1 from Candida rugosa studied by electronic spectroscopies and thermodynamic approaches.

We have used second-order orthogonal designs to obtain empirical models that describe the combined effect of pH and temperature on the secondary structure of a lipase (Lip1) from Candida rusosa. The equations that describe lipase conformational flexibility were derivated from the enzyme alpha helix fraction obtained from the experimental matrix. The thermal unfolding of lipase at different pH values was followed by measuring the circular dichroism signal as a function of temperature over a temperature range of 20-80 °C. The results showed a melting temperature of 58.9 °C at pH 5.5, while at pHs 7.0 and 8.6, the temperature values were 50.2 °C and 36.1 °C respectively. The optimum experimental conditions of conformations with high content of alpha helix were found at high temperature and pH, both at zero time and at one-hour incubation time of enzyme. Important variations in the enzyme secondary structure were induced for the pH and temperature. In contrast, minor changes were observed during the incubation time. This behaviour suggests that the medium pH induces a modification in the enzyme secondary structure and not due to a result of a progressive denaturation process. From the values of thermodynamic functions at different pHs, the system at initial state of unfolding process is previously disordered by the pH effect.

The participation of human serum albumin domains in chemical and thermal unfolding.

Fluorescence spectroscopy and differential scanning calorimetry were used to follow local and global changes in human serum albumin domains during chemical and thermal denaturation of this protein. Results suggests that thermal and chemical treatments involved an unfolding pathway of at least two steps and that domain IIA is not homogeneous. Unfolding at site I exposes a larger hydrophobic area to the solvent than at site II. The bilirubin-binding site showed atypical behavior: a significant increase in the hydrophobic area was exposed to the solvent when its binding site was denatured by guanidine hydrochloride. This result might be due to the high specificity of the bilirubin-binding site, whose binding makes an extensive conformational change in the environment of this site.

Heat treatment of beta-lactoglobulin: structural changes studied by partitioning and fluorescence.

Functional properties of whey protein concentrates (WPC) are primarily dependent on the degree of denaturation of beta-lactoglobulin (beta-LG), the major globular whey protein. Irreversible modifications in the tertiary structure and association state of beta-LG after heat treatment were studied by partition in aqueous two-phase systems and fluorescence quenching. Partitioning of preheated beta-LG in two-phase systems containing 5% (w/w) poly(ethylene glycol) and 7% (w/w) dextran, between pH 6.0 and7.0, are appropriately related with the intensity of heat treatment. An increase in the partition coefficient of beta-LG was observed with increasing temperature of heat treatment. On the other hand, fluorescence quenching of beta-LG by acrylamide was used to study the conformational flexibility of the protein at pH values between 4. 0 and 9.0. The values of bimolecular quenching rate constant (k(q)) obtained showed that beta-LG appears to be more flexible at high pH values, while at low pH the protein assumes a more compact form. The efficiency of acrylamide quenching on preheated beta-LG was substantially more pronounced than for the untreated protein. This difference can be ascribed to the presence of unfolded monomers and aggregates of denatured molecules formed after heat treatment, whose tryptophanyl residues are more exposed to the solvent. In conclusion, the results suggest that partition studies in aqueous two-phase systems and fluorescence quenching are very useful tools to detect changes in conformation and aggregation of beta-LG induced by heat treatment.

Thermal features of the bovine serum albumin unfolding by polyethylene glycols.

Albumin showed very poor affinity for polyethylene glycol molecular weight (Mw) 1000 (30 M(-1)) and Mw 8000 (400 M(-1)) (PEG 1000 and PEG 8000). Polyethylene glycol of low Mw favours the ionization of the tyrosine (TYR) residues of albumin. Such variation might be a consequence of the change in dielectric constant at the domain of the protein by PEG binding. PEGs of high Mws stabilize the native compact state of human albumin showing negative preferential interaction with the protein. Interaction between PEGs and albumin is thermodynamically unfavourable, and becomes even more unfavourable for denatured proteins whose surface areas are larger than those of native ones leading to a stabilization of the unfolded state, which is manifested as a lowering of the thermal transition temperature. PEG 8000 perturbs the structure of the protein surface, partially modifying the layer of water and the microenvironment of the superficial aromatic residues (tryptophan, TRP and TYR) which is in agreement with the modifications of the UV spectrum of albumin by PEG 8000 and circular dichroism (CD) spectrum at high temperatures.

The binding of 3,6-disubstituted bile salts to human serum albumin induces conformational change on the molecule of this protein.

The binding of 3,6-hydroxy and keto disubstituted bile salts to human serum albumin was studied using differential scanning calorimetry, fluorescence spectroscopy and circular dichroism. The bile salts assayed did not produce any modification in the shape of the albumin thermogram, its thermal unfolding process in their presence being reversible; however, an increase in the enthalpy of unfolding and in the Tm was observed in the presence of 3,6-diketo and 3-hydroxy-6-keto bile salts. These two derivatives induced a negative circular dichroism spectrum of the protein around 280-290 nm, quenched the native fluorescence of the buried tryptophan of albumin and induced energy transfer between 1 aniline-8-naphthalene sulfonate and the buried tryptophan 214 of albumin. The presence of a keto group at C6 in the steroid ring of the bile salts plays an important role in producing slight movement of the albumin domains, increasing the distance between domains I and II.

Thermodynamic features of the chemical and thermal denaturations of human serum albumin.

The unfolding process of human serum albumin between pH 5.4 and 9.9 was studied by chemical and thermal denaturations. The experimental results showed that there is no correlation between the stability of albumin at different pH values determined by both methods. The free energy change of unfolding versus concentration of guanidine showed a close dependence on the pH, suggesting that the variation of the electrical charge of albumin influences the final state of the unfolded form of the protein. Spectroscopic techniques, such as native fluorescence of the protein and circular dichroism, demonstrated that the unfolded state of the protein obtained from both methods possesses a different helical content. The solvophobic effect and the entropy of the chains have no influence on the final unfolding state when the protein is unfolded by thermal treatment, while, when the protein is unfolded by chemical denaturants, both effects depend on the medium pH. The results indicate that guanidine and urea interact with albumin by electrostatic forces, yielding a randomly coiled conformation in its unfolded state, while thermal denaturation produces a molten globule state and the aggregation of the protein; therefore, both methods yield different structurally unfolded states of the albumin.

Spectroscopy features of the binding of polyene antibiotics to human serum albumin.

The alteration in the fluorescence spectra observed for the polyene antibiotics nystatin and amphotericin B in the presence of human serum albumin is due to a decrease in the polar character of the antibiotic environment when these are bound to the protein. Amphotericin B showed two types of binding sites, the first having a very high affinity (5.8 x 10(7) M(-1)) and a secondary binding site with an affinity two orders lower than the primary site. This secondary binding site was very sensitive to temperature change. Nystatin yielded only one type of binding site with an affinity of 1.1 x 10(5) M(-1). Nystatin was found to be bound to fatty acid binding sites in albumin, while amphotericin B was not, suggesting that the fatty acid binding sites are not simple, depending on the number of unsaturated bonds on the polyene antibiotic molecule. Both polyene antibiotics displaced bilirubin bound to albumin, which is in agreement with the similarities of the affinity values of this chromophore and the polyene antibiotics with albumin.

Absorption and fluorescence spectra of polyene antibiotics in the presence of human serum albumin.

The alteration in the fluorescence spectra observed for the polyene antibiotics: nystatin and amphotericin B in the presence of human serum albumin is due to a decrease in the polar character of the antibiotic environment when these are bound to the protein. Amphotericin B showed two types of binding sites, the first having very high affinity (5.8 10(7) M(-1]) and a secondary binding site with an affinity one order lower than the primary sites. This secondary binding site was very sensitive to temperature change. Nystatin yielded only one type of binding sites with an affinity of 1.1 10(6) M(-1). An electrostatic component was found in the binding of both ligands, as well as an important disorder at the protein binding sites. However the secondary binding site for AMP showed negative entropic change value, which suggests different mechanism of binding respect to the primary one. Conformational change induced by the temperature in the albumin molecule was detected by nystatin binding. Fatty acids produced an interference in the binding of both antibiotics to albumin.

Destabilization of human serum albumin by polyethylene glycols studied by thermodynamical equilibrium and kinetic approaches.

Both polyethylene glycols (PEG) of MW 8,000 and that of 10000 stabilize the native compact state of human albumin showing negative preferential interaction with the protein. Interaction between these polymers and the protein is thermodynamically unfavorable, and becomes even more unfavorable for denatured protein whose surface areas are larger than those of native ones. PEG of low MW 1000 and 4000 did not show steric exclusion, interacting favorably with hydrophobic side chains made available when the protein was unfolded and leading to a stabilization of the unfolded state, which is manifested as a lowering of the thermal transition temperature. Perturbation of the absorption spectrum of albumin by PEGs confirms that at high temperature the polymers preferentially interact with the denatured state of albumin, but is excluded from the native state at low temperature. This observation is consistent with the fact that PEG is hydrophobic in nature and may interact favorably with the hydrophobic side chain exposed upon unfolding. The lower activation energy for thermal unfolding in the presence of PEG 1000 is in favour of preferential interaction of this polymer with human albumin. PEG of low MW favours the ionization of the tyrosine residues of albumin. It is apparent that pKa decreased with the increase in MW of synthetic polymer. Such variation might be a consequence of the change in dielectric constant at the domain of the protein by PEG binding.

A comparative study of the binding characteristics of ceftriaxone, cefoperazone and cefsulodin to human serum albumin.

The binding to human serum albumin of three cephalosporins of pharmacological interest: cefoperazone, ceftriaxone and cefsulodin was studied by ultrafiltration and differential scanning calorimetry methods. The identification of the binding sites in albumin was also performed using probes for the so-called sites I, II, bilirubin and fatty acids binding sites. Albumin showed two types of binding sites for cefoperazone and ceftriaxone, while for cefsulodin it showed a single type of binding site. The affinity values were: 5.6 10(4) M-1 and 3.1 10(4) M-1 for cefoperazone and ceftriaxone respectively, while cefsulodin showed low affinity (3.8 10(2) M-1). It was found that only cefoperazone interacted in a slight way with site I on serum albumin, while site II and the bilirubin binding site have capacity of binding the three cephalosporins assayed. Ceftriaxone and cefoperazone showed capacity to bind to the fatty acids binding site on albumin. These cephalosporins increased the thermal stability of the protein, suggesting that these ligands are favouring the compact structure of the native form of the protein more than the unfolded form.

Structural features of the hydroxy- and keto-disubstituted bile salts: human serum albumin binding.

The binding of keto- and hydroxy bile salts to human serum albumin, the identity of the bile salts binding sites and the identification of the amino acids present in these sites were studied. The keto bile salts cholanate-3-one (C3), cholanate-3,6-dione (C3-6) and cholanate-3-hydroxy-6-one (KHC) were found to quench the native fluorescence emission of albumin. This suggested that the tryptophan residue of human albumin (residue 214) is accessible to the keto bile salts and not to the hydroxy parent compounds. The binding of the keto bile salts was characterized by a simple population of binding sites with Ka ranging from 22 x 10(4) M-1 for the mono keto bile salt (C3) down to 4 x 10(4) M-1 for the hydroxy-keto bile salt (KHC). The substitution of an oxo group at carbon C3 in C3-6 molecule for a hydroxy group (KHC) produce a significant decreasing of the interaction, suggesting that the hybridization state of the carbon at C3 in the steroid ring of the bile salt molecule is also an essential requirement for bile salts binding. It was found that bile salts are bound to the benzodiazepine binding site on human albumin (site II), producing a perturbation on site I, fatty acids and bilirubin binding site. The presence of only one substituent at C3 (oxo or OH) produce an important perturbation on the fatty acid binding sites, decreasing the polarity of the its microenvironment, while a little effect was observed for the dihydroxy and di-oxo-substituted BS, suggesting that the hydroxy substituents at C6, C7 and C12 do not interact in a significant manner with the fatty acid binding sites on HSA. The participation of specific amino acids in albumin-bile salt binding sites depends on the polar groups on the bile salt molecules as exemplified by the quantitatively different role of lysyl residues like those interacting with KHC, C3 and C3-6, and tyrosyl residue interacting with KHC. The following amino acids in human albumin were found to play a role in the bile salts-albumin interaction: lysyl 195 and 225, several arginyls, histidyl 146 and tyrosyl 411.

The identity of the binding sites of bile salts on bovine serum albumin.

The binding of hydroxy and keto bile salts to bovine serum albumin was studied using probes for the so-called site I, II, bilirubin and fatty acids. Lithocholate, cholanate-3-one and cholanate-3,6-dione produced an interference in the energy transfer process between the albumin-tryptophan residues and the fluorescence markers of sites I and II. The results showed that site II is the binding site for the bile salts on bovine serum albumin. The binding produced a conformational change at site I, bilirubin and fatty acid binding sites, suggesting that these sites may overlap with site II.

Role of electrostatic forces in hydroxy and keto bile salt-albumin interactions: some experimental observations.

Proton binding to bovine serum albumin and effects on hydroxy and keto bile salts-albumin binding were studied within a pH range between 5 and 10. Electrostatic forces contribute to the binding of these ligands to albumin; prototropic groups of albumin such as imidazol are involved in the interaction. Bile salts binding produces a shift in pK of these groups. It is postulated that hydroxy bile salt-albumin binding is linked with the N in equilibrium with B transition of the protein, while for keto bile salts a microarrangement in the protein binding sites is driving the interaction.