The citrate ion increases the conformational stability of alpha(1)-antitrypsin.
Sodium citrate has previously been shown to convert native alpha(1)-antitrypsin into the inactive latent state and cause alpha(1)-antitrypsin to polymerize via the C-sheet pathway instead of the more common A-sheet pathway. In order to begin to understand these dramatic effects, we have examined the influence of low concentrations of sodium citrate upon the structure, stability and function of alpha(1)-antitrypsin. In 0.5 M citrate, the midpoint of guanidine hydrochloride-induced unfolding was increased by 1.8 M and the rate of heat inactivation was decreased approximately 30-fold compared with Tris or phosphate buffer. alpha(1)-Antitrypsin was fully active in the presence of a range of citrate concentrations (0. 1-0.5 M), forming a stable 1:1 complex with chymotrypsin. The association rate constant between alpha(1)-antitrypsin and chymotrypsin was decreased with increasing citrate concentration. Fluorescence and circular dichroism spectroscopy demonstrated no significant changes in the tertiary structure due to the presence of citrate. However, the insertion rate of exogenous reactive-center loop peptide increased with increasing citrate concentration, indicating some structural changes in the A beta-sheet region. Taken together, these data suggest that in the presence of 0.5 M citrate alpha(1)-antitrypsin adopts a highly stable but active conformation.
Bottomley
S P
SP
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. steve.bottomley@med.monash.edu.au
Tew
D J
DJ
D000838:Anions / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D002951:Citrates / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D000077559:Sodium Citrate; D000515:alpha 1-Antitrypsin / Q000737:chemistry*
D000838:Anions; D002951:Citrates; D000515:alpha 1-Antitrypsin; D000077559:Sodium Citrate