pH dependence of specific divalent anion binding to the N-lobe of recombinant human transferrin.
The binding of the two synergistic anion mimics, phosphate and sulfate, and of the synergistic anions, malonate and oxalate, to the N-lobe of recombinant human serum transferrin (hTF/2N) wild-type and H207E mutant protein was assessed by difference ultraviolet (UV) spectroscopy at 246 nm as a function of pH. The absolute values of both the maximum delta epsilon 246 and the Kd decreased with decreasing pH. A plot of -log Kd vs pH gave a straight line with a slope of -1.0. Furthermore, the sum of -log Kd and pH is a constant for each anion binding to each protein. We interpret these data to mean that each anion binds in divalent form along with an H+. The binding equilibrium then appears to be H+ + hTF/2N + X2- reversible K' H-hTF/2N(X) and log K' = -log Kd + pH. A plot of delta epsilon 246 vs pH was sigmoidal with a pKa = 7.4 for both proteins with phosphate and sulfate. When synergistic anions were used with hTF/2N, malonate and oxalate gave pKas of ca. 6.9 and 7.1 for dependence of delta epsilon 246 on pH, but values of 7.3 and 7.6 for the H207E mutant protein. In an attempt to locate the anion binding site in hTF/2N, the binding of sulfate to the single point mutants of the N-lobe of human transferrin, K296E, K296Q, and K206Q, was carried out by difference UV spectroscopy at pH 7.4. In the case of K296E, sulfate binding gave delta epsilon 246 = 0, while for K296Q, it gave a slightly positive delta epsilon 246.(ABSTRACT TRUNCATED AT 250 WORDS)
Cheng
Y
Y
Department of Biochemistry, University of Vermont, Burlington 05405, USA.
Mason
A B
AB
Woodworth
R C
RC
D000818:Animals; D000838:Anions / Q000378:metabolism*; D001665:Binding Sites; D002478:Cells, Cultured; D006224:Cricetinae; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008314:Malonates / Q000378:metabolism; D016296:Mutagenesis; D010070:Oxalates / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013431:Sulfates / Q000378:metabolism; D014168:Transferrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000838:Anions; D008314:Malonates; D010070:Oxalates; D010710:Phosphates; D011994:Recombinant Proteins; D013431:Sulfates; D014168:Transferrin; C030290:malonic acid