Suppression of phase separation in solutions of bovine gamma IV-crystallin by polar modification of the sulfur-containing amino acids.
The calf lens protein gamma IV-crystallin, a strong determinant of the net phase-separation temperature of the lens, was chemically modified with N-bromoacetylethanolamine phosphate. The phase-separation temperatures of solutions of the modified protein were measured and found to be dramatically reduced with respect to those of the native protein. At neutral pH the reagent alkylates only the cysteine and methionine residues and introduces a doubly charged phosphate anion at a maximum distance of 10-12 A from the sulfur atoms. At a protein concentration of 38 g/liter, and with 30% of the cysteines and 40% of the methionines alkylated, the phase-separation temperature is lowered from approximately 25 +/- 2 degrees C to approximately 12 +/- 2 degrees C. The ascending limbs of the coexistence curves for the native and modified proteins were determined at two different degrees of modification. The coexistence curve of the protein with 35% of the cysteines and 40% of the methionines modified shows that as protein concentration approaches the critical concentration of 289 g/liter, there is a much larger suppression of the critical temperature, from approximately 38 +/- 2 degrees C in the native protein to approximately 16 +/- 2 degrees C. Incubation of intact calf lenses in vitro with the reagent results in the suppression of the phase-separation temperature by 3-9 degrees C. These results are consistent with the view that the observed suppression in the critical temperature is due to an increase in the hydrophilicity of the protein in the vicinity of the sulfur-containing residues.
Pande
J
J
Department of Physics, Massachusetts Institute of Technology, Cambridge 02139.
Berland
C
C
Broide
M
M
Ogun
O
O
Melhuish
J
J
Benedek
G
G
D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003459:Crystallins / Q000302:isolation & purification* / Q000378:metabolism; D003545:Cysteine / Q000494:pharmacology*; D007525:Isoelectric Focusing; D007700:Kinetics; D007908:Lens, Crystalline / Q000378:metabolism; D008715:Methionine / Q000494:pharmacology*; D012996:Solutions
D003459:Crystallins; D012996:Solutions; D008715:Methionine; D003545:Cysteine