We study the interaction of 1-anilino-8-naphthalenesulfonate (ANS) with human (HSA) and bovine serum albumin (BSA) by phase and modulation fluorescence spectroscopy. We determined that both HSA and BSA show one or two distinguishable fluorescent sites, depending of the ANS/serum albumin ratio. At above a 1∶1 ANS/HSA molar ratio, the steady-state emission spectra for ANS can be resolved in two components: component 1, emitting with a lifetime (τ1) of 16 ns and a λ1max of 478 nm, with a quantum yield (фf1) of 0.67, and component 2, with a lifetime (τ2) of 2-4 ns and a λ2max of 483 nm, with an average quantum yield (фf2) of about 0.11. Considering these findings, the binding analysis is fitted with a model of two independent sites. Site 1 has an association constantK as1=0.87×10(6)M(-1) and a capacity of 1.04 mol of ANS/mol of HSA, and site 2 aK as2=0.079×10(6)M(-1) and a capacity of 2.34 mol of ANS/mol of HSA. Analysis of fluorescence lifetime distributions shows that the rigidity of the fluorophore environment at site 1 changes when site 2 is occupied. These findings suggest an interconnection between the two sites and that ligands can stabilize the protein's globular structure. To assess the identity of the ANS binding sites we used diazepam as a marker of the site located at the IIIA HSA subdomain and aspirin as a marker of sites located at the IIIA and IIA HSA subdomains. Both ligands displace ANS only from site 1, suggesting that it corresponds to the binding site located at the IIIA sub-domain of the protein. We determined that theK as values for diazepam and aspirin are 0.113× 10(6) and 0.021×10(6) M (-1) respectively.

We investigated how protein changes occur, at the primary or higher structural levels, when proteins are exposed to UV or fluorescent (FL) light while in the complex matrix, milk. Whole milk (WM) or skim milk (SM) samples were exposed to FL or UV light from 0 to 24h at 4°C. Protein oxidation was evaluated by the formation of protein carbonyls (PC), dityrosine bond (DiTyr), and changes in molecular weight (protein fragmentation and polymerization). Oxidative changes in AA residues were measured by PC. Dityrosine and N'-formylkynurenine (NFK), a carbonylation derivative of Trp, were measured by fluorometry. Protein carbonyls increased as a function of irradiation time for both WM and SM. The initial rate for PC formation by exposure to FL light (0.25 or 0.27 nmol/h for WM and SM, respectively) was slower than that following exposure to UV light (1.95 or 1.20 nmol/h, respectively). The time course of NFK formation resembled that of PC. After 24h of UV exposure, SM had significantly higher levels of NFK than did WM. In contrast, WM samples irradiated with UV had higher levels of DiTyr than did SM samples, indicating different molecular pathways. The formation of intra- or intermolecular DiTyr bonds could be indicative of changes in the tertiary structure or oligomerization of proteins. The existence of NFK suggests the occurrence of protein fragmentation. Thus, proteolysis and oligomerization were analyzed by sodium dodecyl sulfate-PAGE. After 24h of exposing WM to UV or FL light, all the proteins were affected by both types of light, as evidenced by loss of material in most of the bands. Aggregates were produced only by UV irradiation. Hydrolysis by pepsin and enzyme-induced coagulation by rennet were performed to evaluate altered biological properties of the oxidized proteins. No effect on pepsin digestion or rennet coagulation was found in irradiated SM or WM. The oxidative status of proteins in milk and dairy products is of interest to the dairy industry and consumers. These findings provide knowledge that could be useful in determining the optimal lighting conditions in the dairy industry in general and in cheese making in particular.

Lipase, protease, and amylase production by Penicillium restrictum in solid-state fermentation was investigated. The basal medium was an industrial waste of babassu oil (Orbignya oleifera) production. It was enriched with peptone, olive oil, and Tween-80. The supplementation positively influenced both enzyme production and fungal growth. Media enriched with Tween-80 provided the highest protease activity (8.6 U/g), whereas those enriched with peptone and olive oil led to the highest lipase (27.8 U/g) and amylase (31.8 U/g) activities, respectively.