Bovine beta-lactoglobulin acts as an acid-resistant drug carrier by exploiting its diverse binding regions.

Binding of fluorine-containing drugs to bovine beta-lactoglobulin, the most abundant whey protein in bovine milk, was investigated by means of (19)F NMR and mass spectrometry. The stoichiometry of the binding and its stability in acidic medium, where beta-lactoglobulin is folded and stable, were also studied, along with competition from molecules that can be regarded as analogs of physiological ligands to bovine beta-lactoglobulin. Conditional binding data were combined with protein structural information derived from circular dichroism and limited proteolysis studies. Spectroscopic techniques were also used to assess whether the bound drugs stabilize the protein structure against denaturation by chaotropes or temperature at various pH values. The results obtained provide evidence for the presence of multiple binding regions on the protein, with a specific and different affinity for structurally different classes of hydrophobic drugs and, more generally, that bovine beta-lactoglobulin can bind and protect against low pH values various classes of drugs of pharmaceutical relevance.

Identification and characterization of a Bowman-Birk inhibitor active towards trypsin but not chymotrypsin in Lupinus albus seeds.

The paper describes the purification, structural characterization and inhibitory properties of a trypsin inhibitor from Lupinus albus L., a leguminous plant believed to be devoid of any protease inhibitor. The protein has been isolated by a newly set-up procedure and characterized by direct amino acid sequencing, MALDI-TOF mass spectroscopy and circular dichroism. Inhibitory properties toward bovine trypsin and chymotrypsin, as well as its thermal and pH stabilities, have been also assessed. The inhibitor is 63 amino acid long (Mr 6858; pI 8.22) and it is capable to inhibit two trypsin molecules simultaneously, with a Kd of 4.2+/-0.4 nM, but not chymotrypsin. BLAST search against UniProtKB/TrEMBL database indicates that the inhibitor belongs to the Bowman-Birk inhibitor (BBI) family. The interest in these serine-protease inhibitors arises from the ability to prevent or suppress carcinogen-induced transformation, as shown in various in vitro and in vivo model systems.

Structural perturbation of alphaB-crystallin by zinc and temperature related to its chaperone-like activity.

alphaB-crystallin is a small heat shock protein that shows chaperone-like activity, as it protects the aggregation of denatured proteins. In this work, the possible relationships between structural characteristics and the biological activity of alphaB-crystallin were investigated on the native protein and on the protein undergoing the separate effects of metal ligation and temperature. The chaperone-like activity of alphaB-crystallin increased in the presence of zinc and when temperature was increased. By using fluorescent probes to monitor hydrophobic surfaces on alphaB-crystallin, it was found that exposed hydrophobic patches on the protein surface increased significantly both in the presence of zinc and when the temperature was raised from 25 to 37 degrees C. The zinc-induced increased exposure of lipophilic residues is in agreement with theoretical calculations performed on 3D-models of monomeric alphaB-crystallin, and may be significant to its increased biological activity.

Bound fatty acids modulate the sensitivity of bovine ß-lactoglobulin to chemical and physical denaturation.

Fatty acids are the natural ligands associated with the bovine milk lipocalin, ß-lactoglobulin (BLG), and were identified by means of mass spectrometry. The naturally bound ligands were found to contribute to the stability of the proteins toward denaturation by both temperature and chaotropes. To assess the nature of the structural regions involved in this stabilization, the thermodynamic and kinetic aspects of the stability of various structural regions of the proteins were studied in the presence of bound palmitate, which is the most abundant natural ligand. Binding of a single palmitate molecule was found to affect not only the stability of the calyx region, where palmitate is bound, but also that of the region at the hydrophobic interface between the barrel itself and the long helix in the protein structure, where the thiol group of Cys121 is buried. This region is known to be essential for the stability of the BLG dimer and is relevant to the generation of "reactive monomers" that are involved in covalent and noncovalent polymerization of BLG and in the formation of covalent adducts with other milk proteins.

Proteolysis of bovine beta-lactoglobulin during thermal treatment in subdenaturing conditions highlights some structural features of the temperature-modified protein and yields fragments with low immunoreactivity.

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 degrees C at neutral pH. At these temperatures beta-lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, beta-lactoglobulin was virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either protease was present during the heat treatment. High-temperature proteolysis occurs in a progressive manner. Mass spectrometry analysis of some large-sized breakdown intermediates formed in the early steps of hydrolysis indicated that both enzymes effectively hydrolyzed some regions of beta-lactoglobulin that were transiently exposed during the physical treatments and that were not accessible in the native protein. The immunochemical properties of the products of beta-lactoglobulin hydrolysis were assessed by using various beta-lactoglobulin-specific antibodies, and most epitopic sites were no longer present after attack of the partially unfolded protein by the two proteases.

Reduction of immunoreactivity of bovine beta-lactoglobulin upon combined physical and proteolytic treatment.

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6.8 for 10 min at 30, 37 and 44 degrees C. The extent of beta-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when chymotrypsin was used. Addition of proteases at ambient pressure to previously pressure-treated beta-lactoglobulin gave only a modest increase in proteolysis with respect to the untreated protein. Products of enzyme hydrolysis under pressure were separated by reverse-phase HPLC, and were found to be different from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact beta-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis under pressure. The results indicate that chymotrypsin effectively hydrolysed hydrophobic regions of beta-lactoglobulin that were transiently exposed during the pressure treatments and that were not accessible in the native protein or in the protein that had been previously pressure treated.