An alternative method to isolate protease and phospholipase A2 toxins from snake venoms based on partitioning of aqueous two-phase systems

Snake venoms are rich sources of active proteins that have been employed in the diagnosis and treatment of health disorders and antivenom therapy. Developing countries demand fast economical downstream processes for the purification of this biomolecule type without requiring sophisticated equipment. We developed an alternative, simple and easy to scale-up method, able to purify simultaneously protease and phospholipase A2 toxins from Bothrops alternatus venom. It comprises a multiple-step partition procedure with polyethylene-glycol/phosphate aqueous two-phase systems followed by a gel filtration chromatographic step. Two single bands in SDS-polyacrylamide gel electrophoresis and increased proteolytic and phospholipase A2 specific activities evidence the homogeneity of the isolated proteins.

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 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.

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.

Thermal stability of human serum albumin by sodium halide salts.

The influence of sodium halide salts at low concentration (above 0.3 M) on the thermal stability of human serum albumin was studied by differential scanning calorimetry. It was found that the sodium halides increase the "melting temperature" and the enthalpy of unfolding of albumin in the following order: F- < Cl- < Br- < I-. It is suggested that the sodium halides do not change the number of solvophobic residues of the protein exposed to the solvent, while an increase of the surface accessible area of the protein to the solvent was found. The increase in the thermal stability of albumin may be due to the preferential hydration of the protein, produced by the structure breaking halides on the water of the bulk solvent.

Lung (agricultural/rural).

Industrialization of farming, animal raising, and forestry has added chemical and mechanical hazards that need to be recognized and prevented. Lung disease among farmworkers can result from a wide variety of hazardous exposures, which include organic dusts, allergens, chemicals, toxic gases, and infectious agents. In addition to nonspecific symptoms of mucous membrane irritation, farmworkers can experience occupational asthma or bronchitis, organic dust toxic syndrome, hypersensitivity pneumonitis, silo filler's disease (toxic hemorrhagic pulmonary edema), and neuromuscular respiratory failure. At risk are farmworkers and those involved in the processing, stocking, transportation, handling, and inspection of unprocessed agricultural, animal, and forestry products; veterinarians; gardeners; game, river, and forest keepers; persons involved in building, supplying, or servicing farm operations; and residents of rural communities. Worker education on the risks of environmental exposures, adherence to safety regulations, and increased knowledge of the cause and prevention of environmental diseases will reduce their prevalence and their adverse human and animal health and socioeconomic effects.

Contribution of the FAD binding site residue tyrosine 308 to the stability of pea ferredoxin-NADP+ oxidoreductase.

The contribution made by tyrosine 308 to the stability of pea ferredoxin-NADP+ reductase was investigated using site-directed mutagenesis. The phenol side chain of the invariant carboxyl terminal tyrosine is stacked coplanar to the isoalloxazine moiety of the FAD cofactor. Fluorescence measurements indicate that this interaction plays a significant role in FAD fluorescent quenching by the reductase apoprotein. Replacement of the tyrosine by tryptophan or phenylalanine caused only a minor increase in the quantum yields of bound FAD, whereas nonaromatic substitutions to serine and glycine resulted in a large fluorescent rise. Results from NADP+ titration experiments support a recent hypothesis [Karplus et al. (1991) Science 251, 60-66], suggesting that the phenol ring of Tyr 308 may fill the nicotinamide binding pocket in the absence of the nucleotide. The stability of the site-directed mutants, judged by thermal- and urea-induced denaturation studies, was lowered with respect to the wild-type enzyme. FNR variants harboring nonaromatic substitutions displayed more extensive destabilization. The decrease in thermodynamic stability correlated with the impairment of catalytic activities [Orellano et al. (1993) J. Biol. Chem 268, 19267-19273]. The results indicate that the presence of the electron-rich aromatic side chain adjacent to the isoalloxazine ring is essential for maximum stabilization of the FNR holoenzyme, resulting in a flavin conformation which optimizes electron flow between the prosthetic group and its redox partners.

Toxic gas inhalation.

Severe toxic fume inhalations, usually accidental due to human error or equipment failure, can result in immediate death from asphyxia or cause mild to severe respiratory distress from acute upper airways inflammation, delayed pulmonary edema, respiratory muscle dysfunction, or a combination of illnesses. Most patients are expected to survive and recover with little or no residual dysfunction regardless of the severity of the initial event. However, in some cases disabling long-term sequelae, eg, bronchiectasis, chronic airflow obstruction, bronchial hyperreactivity, asthma-like disease (reactive airways dysfunction syndrome), bronchiolitis obliterans, or residual psychophysiologic dyspnea, can occur. Therapy of the respiratory effects of irritant gases should follow the general principles used for the treatment of upper and lower airway obstruction, noncardiogenic pulmonary edema, and hemorrhagic pneumonitis while spontaneous healing and recovery occurs, because no specific therapy is available for direct chemical pulmonary injury. Corticosteroids are frequently used and recommended, but their efficacy in altering the course and outcome of respiratory injury has not yet been properly documented.

Hazardous exposure and lung disease among farm workers.

Industrialization of farming, animal raising, and forestry has added new chemical and mechanical hazards that need to be recognized and prevented. Lung disease among farm workers can result from a wide variety of hazardous exposures that include organic dusts, chemicals, and toxic gases. In addition to nonspecific symptoms of mucous membrane irritation, farm workers can develop occupational asthma or bronchitis, organic dust toxic syndrome, hypersensitivity pneumonitis, silo filler's disease (toxic hemorrhagic pulmonary edema), and neuromuscular respiratory failure.

Bile salts-bovine serum albumin binding: spectroscopic and thermodynamic studies.

The binding of hydroxyl and keto bile salts to bovine serum albumin was studied by fluorescence and circular dichroism spectroscopies. It was found that the hydroxyl and keto bile salts produced a quenching of the native fluorescence emission of the protein at 350 nm. In the ligand-protein saturation conditions, cholanate-3-one, cholanate-3,6-dione and beta 5-cholanate produced a 100% fluorescence quenching, while hydroxy bile salts produced only a 50% quenching. This demonstrates that the two tryptophan residues of the protein are accessible to the keto bile salts, while only one tryptophan residue is accessible to the hydroxy parent compounds. Keto bile salts produced a change in the circular is related to a microrearrangement of the environment at the albumin-binding sites. All the tested bile salts produced quenching of the fluorescence probe, 1-aniline-8-naphthalene sulfonate, which is not covalently bound to the protein. This effect is due to an energy transfer between the tryptophan residues and the acceptor fluorescence probe. The binding of hydroxyl bile salts was associated with an endothermic process, while keto bile salts-albumin interaction was associated with a negative enthalpic change.

Thermodynamic study of taurocholate binding to rat serum albumin.

Taurocholate binding to rat serum albumin was studied by equilibrium dialysis. The bile salt-protein interaction was studied under different experimental conditions with respect to temperature; ionic strength; Cl- concentration; pH and the presence of butanol in the medium. The results obtained suggest the existence of two binding sites for taurocholate on the albumin molecule, and indicate that both electrostatic and hydrophobic interaction play a role in the binding process.

Fluorometric studies on the binding of aflatoxin B1 to bovine serum albumin.

The quenching of dansylated BSA fluorescence by Aflatoxin B1 at the dansyl emission peak provided a useful method to study Aflatoxin B1 - BSA interaction, making evident one binding site with hydrophobic characteristics. The Ka = 4.0 X 10(4) M-1 at 18 degrees C could assing to this site a role in Aflatoxin B1 transport, but not in storage in the systemic circulation. Evidence supporting the presence of more binding sites, probably of similar characteristics as the former, was obtained from the study of the displacement of ANS bound to BSA by Aflatoxin B1, in spite of the fact that this interaction cannot be explained as a simple competition between ligands.

Interaction of unconjugated bilirubin with brush border vesicles from proximal tubules of rat kidney.

An interaction between non conjugated bilirubin and brush border vesicles was found which does not change the absorbance spectrum of bilirubin, nor protects it against the action of peroxidase. It seems to reach saturation at about 20 min., and appears to be mainly a superficial binding, probably non specific and relatively weak. The bound amount of bilirubin was high and can not be neglected in any interpretation of the reabsorption of the solute by proximal tubules.

Pulmonary reaction to upper mantle radiation therapy for Hodgkin's disease.

To study the effects of upper mantle radiation therapy on pulmonary function, forced expiratory volume in one second (FEV1), vital capacity (VC), inspiratory capacity (IC), diffusing capacity for CO (DLCO) and diffusion per unit of alveolar volume (DL/VA were determined in 28 patients with Hodgkin's disease, stages 1--3, before therapy and at regular intervals thereafter. Within the first year of follow-up there were significant declines in DLCO, VC, and IC, whereas there were no significant changes in FEV1 or DL/VA. DLCO showed the greatest decline in the largest number of subjects (22/28). Eleven of the 22 had 20 to 60 percent decline of DLCO from baseline. The maximum mean decline in DLCO was -12.7 +/- 3 percent at the 87th +/- 3 days from initiation of therapy postradiation sustained through the 150th day and improving to pretreatment value (+/- 5 percent) by the 8th to 12th month. The changes in DLCO seemed to be independent of the radiation dose ranges evaluated, clinically apparent intrathoracic lymphoma, postradiation radiographic abnormalities and respiratory symptoms. We concluded that impairment in diffusing capacity and loss of vital capacity will develop in most patients receiving upper mantle radiation therapy, indicating that pulmonary reaction occurs despite lung shielding. The functional losses were prolonged and occasionally severe, but were transient and subclinical in most but not all cases. A case of fatal radiation pneumonitis affecting the lung beyond the field of irradiation is reported.