Solution-phase binding of monoclonal antibodies to bee venom phospholipase A2.

The binding of monoclonal anti-bee venom phospholipase A2 antibodies to their antigen was monitored by size-exclusion high performance liquid chromatography. As judged by this panel of six antibodies, honeybee venom phospholipase A2 contains five binding sites, three of which are completely independent epitopes. The study revealed that this PLA2 can accommodate three different antibodies simultaneously. The results demonstrate the utility of size-exclusion high performance liquid chromatography in epitope analyses, such as its ability to compare the relative expansiveness and conformational state of the epitopes and to enumerate the antibodies that the antigen can accommodate simultaneously. The data provide compelling evidence that one of the monoclonal antibodies, M5 (which activates the enzyme), recognizes a different conformation of phospholipase A2 than do the other antibodies. The results also demonstrate that different pairs of monoclonal antibodies differ in their predilection to form high molecular weight complexes with the antigen.

Pathogenic potential of human monoclonal immunoglobulin light chains: relationship of in vitro aggregation to in vivo organ deposition.

The deposition of certain Bence Jones proteins as tubular casts, basement membrane precipitates, or amyloid fibrils results in the human light-chain-associated renal and systemic diseases--myeloma (cast) nephropathy, light-chain deposition disease, and immunocyte-derived (primary or AL) amyloidosis. To determine if light-chain nephrotoxicity or amyloidogenicity is related to the propensity of these components to form high molecular weight aggregates under physiological conditions, we used a size-exclusion chromatographic system to study 40 different Bence Jones proteins. Each samples was tested over a wide range of protein concentration in three different buffers varying in pH, osmolality, and the presence or absence of low concentrations of urea. Thirty-three of the 35 proteins found clinically and/or experimentally to form in vivo pathologic light-chain deposits were shown to undergo high-order self-association and form high molecular weight aggregates. In contrast, of five nonpathologic proteins, one showed polymerization under the chromatographic conditions used. The correlation between the in vivo results achieved by size-exclusion chromatography and that found in vivo provides (i) a rapid diagnostic method to identify potential nephrotoxic or amyloidogenic Bence Jones proteins and (ii) an experimental means to gain new insight into the physicochemical basis of light-chain aggregation and the treatment of those invariably fatal disorders associated with pathologic light-chain deposition.

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.