Reversible inactivation of serpins at acidic pH.
The inhibitory activity of the serpins alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, alpha(2)-antiplasmin, antithrombin and C(1)-esterase inactivator is rapidly lost at pH 3 but slowly recovers at pH 7.4 with variable first-order rates (t(1/2)=1.4-19.2 min). All except alpha(1)-antichymotrypsin undergo a variation in intrinsic fluorescence intensity upon acidification (midpoint ca. 4.5) with a slow bi-exponential return to the initial intensity at pH 7.4 (mean t(1/2)=2.3-23 min). No correlation was found between the time of fluorescence recovery and that of reactivation. The acid-treated serpins are proteolyzed at neutral pH by their target proteinases. alpha(1)-Proteinase inhibitor was studied in more detail. Its acidification at pH 3 has a mild effect on its secondary structure, strongly disorders its tertiary structure, changes the microenvironment of Cys(232) and causes a very fast change in ellipticity at 225 nm (t(1/2)=1.6s). Neutralization of the acid-treated alpha(1)-proteinase inhibitor is an exothermic phenomenon. It leads to a much faster recovery of activity (t(1/2)=4+/-1 min) than of fluorescence intensity (t(1/2)=23+/-19 min), ellipticity (t(1/2)=32+/-4 min) and change in total energy, indicating that the inhibitory activity of alpha(1)-proteinase inhibitor does not require a fully native structure.
Boudier
Christian
C
CNRS UMR 7175, Département Physicochimie et Pharmacochimie des Interactions Moléculaires et Cellulaires, Faculté de Pharmacie, Université Louis Pasteur, Strasbourg I, F-67401, Illkirch, France. christian.boudier@pharma.u-strasbg.fr
Bousquet
Jean-Alain
JA
Schauinger
Sébastien
S
Michels
Bernard
B
Bieth
Joseph G
JG
D000991:Antithrombins / Q000737:chemistry; D003174:Complement C1 Inactivator Proteins / Q000737:chemistry; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D000514:alpha 1-Antichymotrypsin / Q000737:chemistry; D000515:alpha 1-Antitrypsin / Q000737:chemistry; D000979:alpha-2-Antiplasmin / Q000737:chemistry
D000991:Antithrombins; D003174:Complement C1 Inactivator Proteins; D015843:Serpins; D000514:alpha 1-Antichymotrypsin; D000515:alpha 1-Antitrypsin; D000979:alpha-2-Antiplasmin