Investigation of zinc binding metallothioneins' polymerization in tris(hydroxymethyl)-aminomethane buffer by coupling of size exclusion chromatography with electrospray ionization mass spectrometry.
Polymerization of metallothioneins (MTs) is one of the commonly encountered puzzles in researching the structure and function of metallothioneins. In this work, a method involving SEC coupled with negative ion electrospray ionization mass spectrometry (ESI-MS) detection has been developed for the study of zinc binding MTs' polymerization in tris(hydroxymethyl)-aminomethane (TRIS) acetate buffer at physiological pH. This hyphenated technique allows separating the different polymeric states of MTs by SEC, followed by on-line identification of the individual MT subisoforms in each polymeric peak by ESI-MS detection. Purified MT subisoforms (MT-2d and MT-2a), MT-2d and MT-2a mixture and rabbit liver MT complexes were investigated in the experiments to confirm the results obtained. From the results, both oxidative polymerization and non-oxidative oligomerization were found. The cystein-dependent oxidation results in the tetrameric peak as shown in the chromatograms of oxidized MT-2d, and stable dimeric and monomeric of MT were detected in this peak by MS. For the dimeric and trimeric peaks, different MT subisoforms were detected. In the five major subisoforms detected in rabbit liver MT complexes, MT-2a and MT-2c exist primarily as trimer, while MT-2e, MT-2d and MT-1a exist mainly as dimer. Our results suggest that in the three kinds of polymers, dimer, trimer and tetramer that were found in samples, the tetramer comes from the oxidation of MT molecular; for the dimer and trimer resulting from cystein independent oligomerization, they are closely associated with the charge of subisoform.
Shen
Jin-Can
JC
Department of Chemistry and the Key Laboratory of Analytical Sciences of the Ministry of Education, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China; Inspection Center of Import and Export Food, Shenzhen Entry-Exit Inspection and Quarantine Bureau of PRC, Shenzhen 518067, China.
Liu
Jie
J
Zhuang
Zhi-Xia
ZX
Wang
Xiao-Ru
XR
Lee
Frank S C
FS