Chemical modification of buried lysine residues of bovine serum albumin and its influence on protein conformation and bilirubin binding.
Using a double modification technique about 20% of the lysine residues of bovine serum albumin (BSA) which are not easily accessible in the native protein have been modified. The technique involved approximately 80% modification of lysine residues of BSA with citraconic anhydride followed by chemical modification of the remaining lysine residues with acetic anhydride, succinic anhydride, potassium cyanate, or O-methylisourea. Finally, these preparations were decitraconylated under mild acidic conditions to yield acetylated, succinylated, carbomylated or guanidinated BSA. All of these preparations were found to be homogeneous with respect to charge and size. The spectral, hydrodynamic and bilirubin binding properties of these preparations are described. In contrast to most of the highly modified proteins these preparations with the exception of succinylated BSA are very similar to native BSA in their spectral and hydrodynamic properties. However, the equilibrium association constant (Ka) with bilirubin measured by fluorescence quenching was decreased by about 100-fold in acetylated, carbamylated and succinylated BSA, but only 3-fold in guanidinated BSA. Since conformationally acetylated and carbamylated BSAs are identical to guanidinated BSA we conclude that the decrease in Ka in these preparations is solely due to loss of positive charge on 'critical' lysine residues. The results support a binding model for BSA in which bilirubin binding site is buried and the protein undergoes a series of relaxational changes in conformation upon interaction with bilirubin.
Mir
M M
MM
Department of Biochemistry, J.N. Medical College, A.M.U., Aligarh, India.
Fazili
K M
KM
Abul Qasim
M
M
D000818:Animals; D001663:Bilirubin / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D005453:Fluorescence; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis
D012710:Serum Albumin, Bovine; D008239:Lysine; D001663:Bilirubin