[Spectrophotometric studies on the binding of Victoria Blue 4R to oxidized insulin]

The empirical demonstration of insulin by the basic (cationic) dye Victoria Blue 4R (V4R; Ivić 1959) shows the character of a histochemical reaction (Wohlrab et al. 1985), while the chemistry+ of the reaction is not quite understood yet. Aim of this investigation: Will the spectral behaviour of V4R in visible light be influenced by oxidized or non-oxidized insulin? Dependent on the concentration, V4R shows in aqueous solution 2 absorption maxima (lambda = 597 and 558 nm), which represents monomers and dimers of the dye. With increasing dye concentration (200 mumol/l), V4R forms dimers and even higher polymers. Constant V4R concentration (200 mumol/l) results with increasing concentration of oxidized insulin (4 to 16 mumol/l) in a reduction of extinction, while the extinction at lambda = 558 nm (dimers) is more decreased than at lambda = 597 nm (monomers). Non-oxidized insulin has no remarkable influence on the absorption behaviour of V4R. Extinction measurements on V4R stained B-cells of islets of Langerhans after pre-oxidation of the section resulted in a main absorption maximum at lambda = 554 nm.

Depending on the concentration, the dye V4R is associated with the oxidized insulin in aqueous solution, which is also indicated by the occurrence of an isobestic point in the curve behaviour. This is expressed by the establishment of a concentration-dependent equilibrium between the dye V4R and the oxidized insulin (change in the dissociation and aggregation behaviour respectively of the dye V4R). The determined main absorption maximum (lambda = 554 nm) in the biological material points in the same way to interactions between the stain and oxidized insulin.