Interactions between carbonic anhydrase and its inhibitors revealed by gel electrophoresis and circular dichroism.
Structural properties, and especially the differential stability, of complexes between carbonic anhydrase (CA) and three sulfonamide inhibitors, acetazolamide, dorzolamide and methazolamide, were investigated by spectroscopic and electrophoretic techniques. These included denaturant gradient gel electrophoresis either across a urea or a steady-state transverse sodium dodecyl sulfate (SDS) gradient. Acetazolamide, the smallest and most hydrophilic of the sulfonamides, forms the most stable complex in the presence of urea, whereas dorzolamide, with a bulky and hydrophobic structure, is most stable against the effects of SDS. At pH 7.4, complexes with dorzolamide show minimal changes in mobility across the SDS gradient, as if unaffected by the detergent, both in the presence and in the absence of excess ligand in the gel. When bound to both acetazolamide and methazolamide, on the other hand, CA displays an increase in mobility above 0.05% SDS, lower in the presence than in the absence of excess ligand. The finding of a distinct pattern for the unliganded enzyme, however, suggests the complexes can still retain the ligand, although binding of the surfactant changes their charge density. Under saturating conditions and in the presence of SDS, the surface charge of all complexes is much lower than for unliganded, denatured CA. Circular dichroism (CD) spectra clearly indicate that the increase in secondary structure and the decrease in tertiary structure brought about in CA by the presence of low concentrations of SDS are largely prevented by complexing with the inhibitors. These observations point out peculiar properties of each CA inhibitor, of potential value in the definition of their biological activities and also in the potential development of novel antagonist molecules.
Gianazza
E
E
Istituto di Scienze Farmacologiche, Università di Milano, Italy. elisabetta.gianazza@unimi.it
Sirtori
C R
CR
Castiglioni
S
S
Eberini
I
I
Chrambach
A
A
Rondanini
A
A
Vecchio
G
G
D000086:Acetazolamide / Q000378:metabolism*; D000818:Animals; D002257:Carbonic Anhydrase Inhibitors / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007525:Isoelectric Focusing; D008704:Methazolamide / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D013449:Sulfonamides / Q000378:metabolism*; D013876:Thiophenes / Q000378:metabolism*; D017186:Titrimetry; D014508:Urea
D002257:Carbonic Anhydrase Inhibitors; D013449:Sulfonamides; D013876:Thiophenes; D012967:Sodium Dodecyl Sulfate; D014508:Urea; C062765:dorzolamide; D002256:Carbonic Anhydrases; D000086:Acetazolamide; D008704:Methazolamide