Effects of pH and calcium ion on self-association properties of two dimeric phospholipases A2.
The monomer-dimer equilibria of the dimeric phospholipases A2 from Crotalus atrox and Agkistrodon piscivorus piscivorus venoms were examined chromatographically as a function of pH and in the presence versus absence of the essential cofactor, calcium ion. At neutral pH without calcium, the subunits of both enzymes reequilibrated sufficiently slowly that dimer and monomer were separated by size exclusion chromatography. At pH 4.2 and lower, the dimers underwent rapid dissociation and reassociation, eluting as single broad peaks whose position as a function of applied protein concentration could be analyzed to determine association constants using an algorithm that estimates these values based on elution positions. Lowering the pH from 7.0 to 4.2 increased the self-association constant of the C. atrox enzyme by 1 order of magnitude and that of the A. p. piscivorus dimer by a factor of 3. Calcium ion, an essential cofactor of phospholipase A2, converted the kinetic behavior of the dimers at neutral pH from slow to virtually instantaneous on the time scale of the chromatography runs, 40 min. Calcium ion also altered the thermodynamic stability of the enzymes; the association constant of A. p. piscivorus phospholipase A2 in neutral pH buffer was reduced by approximately 2 orders of magnitude, whereas that of C. atrox was increased by a factor of 6. The structural basis for the disparate effects of calcium ion on these two acidic, dimeric venom phospholipases A2 is uncertain. This study illustrates the importance of calcium ion and pH on the solution behavior of the dimeric members of this class of enzymes.
Myatt
E A
EA
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Stevens
F J
FJ
Sigler
P B
PB
D002118:Calcium / Q000502:physiology*; D003435:Crotalid Venoms; D006863:Hydrogen-Ion Concentration; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D054467:Phospholipases A2
D003435:Crotalid Venoms; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium