A time-dependent, two-step binding mode of the nitro dye flavianic acid totrypsin in acid media

Synthetic dyes bind to proteins causing selective coprecipitation of the co mplexes in acid aqueous solution by a process of reversible denaturation th at can be used as an alternative method for protein fractionation. The even ts that occur before precipitation were investigated by equilibrium dialysi s using bovine trypsin and flavianic acid as a model able to cause coprecip itation. A two-step mode of interaction was found to be dependent on the in cubation periods allowed for binding, with pronounced binding occurring aft er 42 h of incubation. The first step seems to involve hydration effects an d conformational changes induced by binding of the first dye molecule, foll owing rapid denaturation due to the binding of six additional flavianate an ions to the macromolecule.