Purification and characterization of human alpha 2-macroglobulin conformational variants by non-ideal high performance size-exclusion chromatography

摘要: Human alpha 2-macroglobulin (alpha 2M) was eluted as a single nondispersed peak from TSK-G4000SW size exclusion chromatography column equilibrated in 20 mM-sodium phosphate/100 mM-NaCl, pH 7.2 (PBS). The void volume and total accessible of the were 6.08 ml 14.42 ml. elution (Ve) native 2M 9.20 +/- 0.04 Ve altered minimally by changing ionic strength or adding ethanol to equilibration buffer. Ternary 2M-trypsin, containing 2 mol proteinase/mol inhibitor, 2M-methylamine failed be well-defined peaks when PBS. majority either preparation recovered slowly at values greater than 14.5 ml, reflecting significant nonideal interactions with support structure. Addition 10% increased buffer independently caused form reacted distinct decreased Ve, suggesting that included hydrophobic electrostatic adsorption. When 80 phosphate/320 7.2, partial resolution ternary 2M-trypsin obtained run. this 13.15 0.08 11.94 0.14 respectively. 8.84 0.03 resolving capacity exploited purify rapidly efficiently solutions contained amounts binary (1 inhibitor). This purification complete within limits sensitivity denaturing nondenaturing polyacrylamide-gel electrophoresis. 2M-plasmin well resolved 2M. 12.88 0.32 7.2. A number procedures used prepare up 90% 2M-trypsin. these various intermediate between conformations complex, reflected mobility electrophoresis, identical, confirming previous results. Finally, trypsin cleaved more two, many all four subunits.