ObjectiveTo separate and identify the characteristics of testicular metal-binding proteins (TMBPs) induced by exhaustive swimming in rats. MethodsEight male SD rats were subjected to a bout of exhaustive swimming (8 for control) and the testes and liver were taken out 6 hours after the exercise. TMBPs and hepatic metallothionein (MT) were determined by cadmium-saturation assay. The extracts from the testes and liver by cadmium-saturation method were separated by tricine-SDS-PAGE and TMBPs protein bands were identified by LC-MS-MS. ResultsThe results of cadmium-saturation assay showed that the TMBPs content was significantly increased in the exhaustive swimming rats [(113.71±11.72)nmol Cd/g testis] than that in the controls [(87.14±12.72)nmol Cd/g testis] (P<0.01), and the hepatic MT was (64.70±14.89)μg/g, significantly higher than the (7.32±3.31)μg/g in the controls (P<0.001). Ubiquitin, superoxide dismutase [Cu-Zn](Cu-Zn-SOD), casein-like phosphoprotein (CLPH) were identified in TMBPs, and MT was also included. ConclusionsTMBPs are mainly composed of ubiquitin, SOD and CLPH, with characteristics of metal binding, heat resistance and induction. The cadmium-saturation assay is not a specific method for MT determination.