2-anilinonaphthalene-6-sulfonic-acid and 5-5--bis(8-(phenylamino)-1-naphthalenesulfonate)

1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and 2-(p-toluidino)naphthalene-6-sulfonic acid (2,6-TNS) were evaluated as additives in different buffers for the detection of bovine whey proteins using laser-induced fluorescence (LIF) monitoring in capillary electrophoresis (CE). These N-arylaminonaphthalene sulfonates furnish a large fluorescence emission when associated to some proteins whereas their emission in aqueous buffers, such as those used in CE separations, is very small. To select the best detection conditions, the fluorescence of these probes was first compared using experiments carried out in a fluorescence spectrophotometer. Using bovine serum albumin (BSA) as a model protein, it was demonstrated that 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 8 and pH 10.2) and the fluorescent probe 2,6-TNS gave rise to the highest increase in fluorescence for BSA. When the composition of these separation buffers was optimized for the electrophoretic separations, CHES buffer, pH 10.2 was chosen as the most suitable buffer to detect bovine whey proteins. The limit of detection obtained for some whey proteins in CE separations was about 6.10(-8) M for BSA, 3.10(-7) M for beta-lactoglobulin A (beta-LGA), 3.10(-7) M for beta-lactoglobulin B (beta-LGB), and 3.10(-6) M for alpha-lactalbumin (alpha-LA). These detection limits were compared to those achieved using UV detection under the same separation conditions. The results showed that the detection limits of BSA, beta-LGA and beta-LGB were twice as good using LIF than with UV detection. However, the limit of detection for alpha-LA was better when UV was used. The applicability of LIF detection to CE separation of whey proteins in bovine milk samples was also demonstrated.

Topics: Anilino Naphthalenesulfonates; Animals; Cattle; Electrophoresis, Capillary; Fluorescence; Fluorescent Dyes; Hydrogen-Ion Concentration; Indicators and Reagents; Lactalbumin; Lactoglobulins; Lasers; Milk Proteins; Serum Albumin, Bovine; Whey Proteins

We have previously found that the sulfhydryl groups of tubulin are sensitive reporters of the effects of ligands on the tubulin molecule. In this study, we examined the effects of three anilinonaphthalenesulfonates on the interaction of tubulin with iodo[14C]acetamide and N, N'-ethylenebis(iodoacetamide). We found that 1,8-anilinonapthalensulfonate (1,8-ANS) and 2,6-anilinonaphthalenesulfonate (2,6-ANS) had no effect on the reaction with iodo[14C]acetamide. In contrast, bis(1,8-anilinonaphthalenesulfonate) (BisANS), an inhibitor of microtubule assembly, had a complex effect. Low concentrations of BisANS, where presumably only the high-affinity binding site was saturated, had little or no effect on alkylation. Higher concentrations of BisANS caused a strong enhancement of alkylation. None of these compounds had any effect on the reaction with N,N-ethylenebis(iodoacetamide). Our results suggest that the binding of BisANS, 2,6-ANS and 1,8-ANS to tubulin is complex and very different from that of the other anti-tubulin drugs. The correlation between the effects of drugs on alkylation of tubulin and the binding of BisANS is consistent with a model whereby the alkylatable sulfhydryls are located in apolar regions of the tubulin molecule.

Topics: Alkylation; Anilino Naphthalenesulfonates; Iodoacetamide; Protein Binding; Tubulin