Guillermo Picó

Thermodynamic features of the thermal unfolding of human serum albumin

The unfolding process of human serum albumin (HSA) was studied by thermal effect on the native fluorescence of the protein, thermal inactivation of the hydrolase activity of albumin and differential scanning calorimetry using the high sensitive calorimeter developed by Privalov. The denaturation process can be described by an approximation of the model of Eyring and Lumry: native [symbol: see text] unfolded reversible [symbol: see text] unfolded irreversible. It was found that the rate of irreversible step was very slow (at temperatures below 74 degrees C), allowing the resolution of the denaturation process as a reversible one on the basis of two states approximation. However, the presence of intramolecular cooperation in the thermal denaturation process at temperatures above 74 degrees C cannot be discarded, which might be favoring the aggregation of albumin molecules. The midpoint temperature of unfolding obtained by differential scanning calorimetry was of 63.1 degrees C +/- 0.4 at pH 7.4. This value was independent of the rate of scanning and it is in agreement with those obtained by techniques such as thermal effect on the protein fluorescence and on the hydrolase activity of albumin. The enthalpy of unfolding at pH 7.4 was 88.9 +/- 4 Kcal/mol. This value was low compared with those obtained for other proteins, suggesting the presence of a molten globule in the unfolding pathway of albumin. The neutral-basic conformational change (pH 7.4) of albumin did not modify the thermal stability and the enthalpy of denaturation of the protein. A pH below 4.3 (transition acid-neutral) the presence of a second peak in the thermogram of albumin with a TM of 46.2 degrees C +/- 0.9 would be suggesting a lost of cooperativity between the various domains of albumin in the unfolding.

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.

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.

Comparison between the thermodynamic features of α1-antitrypsin and human albumin partitioning in aqueous two-phase systems of polyethyleneglycol-dextran

The partitioning features of human serum albumin and alpha1-antitrypsin in aqueous two-phase systems of dextran and polyethyleneglycol were studied. The effect of factors that affect the electrostatic term of Albertsson equation such as pH, ionic strength, presence of neutral salts as well as those which affect the non-electrostatic term such as polyethyleneglycol mol. wt. and temperature were assayed. At room temperature, the positive entropy and enthalpy changes associated to the partition may be due to a release of part of the structured water in the domain of proteins caused by H-bonds rupture when the proteins are transferred to the upper phase. This behaviour may be explained on the basis of a preferential hydration of the proteins in presence of dextran (bottom phase) and a preferential interaction of polyethyleneglycols with the protein domain (top phase). The electrostatic interactions were similar for both proteins due to the proximity of their isoelectric point and similar dissociation profiles of their prototropic groups.

Partition in aqueous two-phase system: Its application in downstream processing of tannase from Aspergillus niger

Tannase from Aspergillus niger was partitioned in aqueous two-phase systems composed by polyethyleneglycol of molar mass 400, 600 and 1000 and potassium phosphate. Tannase was found to be partitioned toward the salt-rich phase in all systems, with partition coefficients lower than 0.5. Partition coefficients values and low entropic and enthalpic changes associated with tannase partition suggest that the entropic effect may be the driving force of the concentration of the enzyme in the bottom phase due to the high molar mass of the enzyme. The process was significantly influenced by the top phase/bottom phase volume ratio. When the fungal culture broth was partitioned in these systems, a good performance was found, since the enzyme recovery in the bottom phase of the system composed by polyethyleneglycol 1000 was around 96% with a 7.0-fold increase in purity.

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.

A comparative study of the binding characteristics of ceftriaxone, cefoperazone and cefsudolin 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: 5.6 104 M‐1 and 3.1 104 M‐1 for cefoperazone and ceftriaxone respectively, while cefsulodin showed low affinity (3.8 102 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.

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 (5.8 107 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 1.1 106 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 entropie change value, which suggests different mechanim 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.

Continuous method to determine the trypsin inhibitor activity in soybean flour.

The determination of trypsin inhibitor (TI) activity is of importance to evaluate the nutritional value of soybean flours. An analytical method, which involves a continuous spectrophotometric rate determination for trypsin activity against the substrate N-benzoyl-DL-arginine p-nitroanilide, is proposed as an alternative to the standard discontinuous assay. Stopping the reaction with acetic acid and a centrifugation/filtration step to decrease turbidity are not required, thus reducing costs and sample preparation time. The TI activity of different flour samples, determined by both assays, demonstrated to be statistically comparable, irrespective of the TI concentration level. The coefficients of variation of the novel method did not exceed 8% at any concentration level. The curves of progress reaction showed a non-linear behavior in samples without TI. A reduction of incubation time from 10min to 2min increased the method sensitivity and extended its linear range. A more economical, faster and simpler assay was developed.

Comparison of soybean hull pre-treatments to obtain cellulose and chemical derivatives: Physical chemistry characterization

The cellulose from soybean hull, a waste without value from the argentine agriculture, was successfully obtained by using two different treatments: the traditional alkaline-bleaching pathway and from a simple pre-alkaline treatment at low temperatures. The comparison of both methods yielded similar results regarding its ability to open the lignin cellulosic structure of the hull and the total elimination of the lignin content. Fourier Transform Infrared spectroscopy (FT-IR), Thermogravimetric analysis (TGA), Scanning electron microscopy (SEM), 13C nuclear magnetic resonance (13C-RMN) and Raman spectroscopy were used to characterize the structures and the properties of cellulose. The results showed that cellulose can be easily obtained with just an alkaline pre-treatment of 5% (w/v) NaOH during 40u202fh at 50u202f°C and free of any lignin content. The attachment of different functional groups, such as -COOH and (CH3)3N+, changed the physicochemical properties of the obtained cellulose, showing mayor crystalline structure, and consequently modifying the swelling capacity and its ability to adsorb model proteins.