Biochemical characterization of monomeric and dimeric cadmium-substituted metallothionein-3

Summary

Human brain-specific metallothionein (MT-3) has been expressed in E. coli in the presence of cadmium salt and purification of protein combining ethanol precipitation, gel filtration and ionic exchange chromatography has been elaborated. Results obtained demonstrate the occurrence of protein in distinct metastable monomeric and dimeric forms which could be successfully separated by ionic exchange chromatography. Monomeric and dimeric MT-3 has been characterized by UV, CD spectroscopy as well as by sulphydryl content. UV spectrum of monomeric form is similar to the spectrum of common Cd₇MT-2 but exposes slightly lower extinction coefficent. In CD spectrum of monomeric MT-3 the lowest energy transition is blue-shifted (maximum at 254 nm) as compared to Cd₇MT-2 (maximum at 259 nm) and CD band at 225 nm is negative. Dimeric MT-3 exposes in UV spectrum a shoulder at 250 nm and a new transition with maximum at 285 nm. CD spectrum of dimeric MT-3 exposes positive band at 259 nm and negative band at 230 nm and broad shoulder in region up to 300 nm. CD band at 225 nm is negative. Dimeric MT-3 dissociates into monomeric form by spontaneous incubation as well as by the action of sulphydryl reagents (2-mercaptoethanol and dithiothreitol) pointing towards existence of intermolecular disulphide bridges in dimeric MT-3.