Suppression of phase separation in bovine gamma IV crystallin solutions: effect of modification by charged versus uncharged polar groups.
gamma IVa Crystallin, the ocular lens protein with the highest critical temperature (Tc) for phase separation, has been chemically modified with N-ethylmaleimide (NEM) at neutral pH. NEM, a polar but uncharged modifier reacts with the cysteine residues of the protein. A maximum of 80-85% of the thiol groups are modified and the phase separation temperature is lowered by about 10 degrees C at a protein concentration of 30 mg ml-1. The coexistence curve of the NEM-modified protein with nearly four out of the six cysteines modified was determined and compared with that obtained by modifying the protein with a charged reagent, N-bromoacetylethanolamine phosphate (NBAEP). NBAEP modifies both cysteine and methionine residues of gamma IVa crystallin. The results of these two modification studies indicate that for the same total degree of modification of the sulfur-containing residues, the suppression in Tc due to the charged NBAEP is at least twice as large as that due to the polar but uncharged NEM. In order to obtain some measure of the relative hydrophilicities of the two modifiers, we have estimated the free energies of hydration of the nonionic segments of NEM and NBAEP, using structural additivity schemes. We find that the intrinsic hydrophilicities of the nonionic segments of these two modifiers are nearly equal. Hence the much larger suppression of Tc resulting from NBAEP modification is most probably due to the strongly polar, doubly charged phosphate group.
Pande
J
J
Center for Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge 02139.
Ogun
O
O
Nath
C
C
Benedek
G
G
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002386:Cataract / Q000517:prevention & control; D002417:Cattle; D003459:Crystallins / Q000032:analysis / Q000187:drug effects; D004983:Ethanolamines / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D011489:Protein Denaturation; D013696:Temperature; D013997:Time Factors
D000596:Amino Acids; D003459:Crystallins; D004983:Ethanolamines; C020161:N-bromoacetylethanolamine phosphate; D005033:Ethylmaleimide