Explanation for the high heat stability of thyroxine binding globulin-Chicago.
OBJECTIVE
Thyroxine binding globulin-Chicago (TBG-Chicago), a variant of TBG with enhanced heat stability, was isolated at the University of Chicago from a 22 year old subject of an African American lineage. High thermodynamic stability in serine proteinase inhibitors (serpin) is the hallmark of reactive center loop (RCL) inserted conformation and this aspect has not been explored in TBG-Chicago, a serpin molecule. It is hypothesised that the high heat stability of TBG-Chicago is due its loop inserted state.


METHODS
Recombinant (r) TBG-Chicago and normal (r) TBG were expressed in baculovirus system. Stability of the freshly made proteins was assessed on Native- PAGE by: 1. heating at 65 degrees C/30 min; 2. incubating at 37 degrees C/24 h. Susceptibility of the RCL of both the TBG's to endoproteinase cleavage and the ability to accept synthetic RCL mimetic peptide were assessed before and after incubation at 37 degrees C /24 h (SDS-PAGE/ Native PAGE).


RESULTS
It was found that rTBG-Chicago aggregates at 65 degrees C, accepts RCL mimetic peptide and is cleaved by endoproteinase. In contrast, rTBG-Chicago that had been incubated at 37 degrees C/24 h showed enhanced heat stability at 65 degrees C, reduced ability to accept synthetic peptide and decreased susceptibility to endoproteinase cleavage (besides having changed mobility on Native gel).


CONCLUSIONS
The results support the conclusion that freshly isolated TBG-Chicago exists in loop expelled conformation. However at 37 oC, the protein readily converts to a more stable loop inserted conformation and could explain why plasma TBG-Chicago was found to have enhanced heat stability in the 22 year old subject.
Duhan
U
U
Department of Oral Medicine and Diagnostic Sciences, College of Dentistry, University of Illinois at Chicago, Chicago, IL 60612, USA.
Patston
P
P
D016367:Baculoviridae / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006358:Hot Temperature; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry; D013975:Thyroxine-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D055815:Young Adult
D010455:Peptides; D011994:Recombinant Proteins; D013975:Thyroxine-Binding Proteins; C050338:thyroxine-binding globulin Chicago; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N