The conformational state of human alpha 2-macroglobulin influences its dissociation into half-molecules by sodium thiocyanate.
Sodium thiocyanate dissociates native human alpha 2-macroglobulin into half-molecules consisting of two disulfide-bonded subunits, when the salt concentration is equal to or exceeds 1.2 M. Incubation with 1.6 M sodium thiocyanate for 1 h at 22 degrees C dissociates about 90% of alpha 2-macroglobulin into half-molecules. The half-molecules remain stable when the concentration of sodium thiocyanate is reduced to 0.2 M or zero, demonstrating that reassociation does not occur under these conditions. The internal thiol esters of the half-molecules are intact because they can be exposed by treatment with methylamine or trypsin. The noncovalent interaction between the disulfide-bonded dimers is stronger in the "closed-trap" than in the "open-trap" conformation of alpha 2-macroglobulin. The cleavage in the bait region by trypsin makes alpha 2-macroglobulin completely stable toward dissociation, and alpha 2-macroglobulin remains in a tetrameric state in 2.2 M sodium thiocyanate even when trypsin is not covalently bound to it. The increase in fluorescence with time indicates that conformational changes occur as a consequence of dissociation.
Shanbhag
V P
VP
Department of Biochemistry, Umeå University, Sweden.
Stigbrand
T
T
Jensen
P E
PE
D006801:Humans; D066298:In Vitro Techniques; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives; D008744:Methylamines; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates / Q000494:pharmacology*; D014357:Trypsin; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000187:drug effects*
D008744:Methylamines; D013861:Thiocyanates; D000511:alpha-Macroglobulins; C014674:methyl methanethiosulfonate; C024553:sodium thiocyanate; D008741:Methyl Methanesulfonate; C027451:methylamine; D014357:Trypsin