The binding of pyridoxal 5'-phosphate to human serum albumin.
Most of the pyridoxal 5'-phosphate (PLP) in plasma is bound to protein, primarily albumin. Binding to protein is probably important in transporting PLP in the circulation and in regulating its metabolism. The binding of PLP to human serum albumin (HSA) was studied using absorption spectral analysis, equilibrium dialysis, and inhibition studies. The kinetics of the changes in the spectrum of PLP when mixed with an equimolar concentration of HSA at pH 7.4 followed a model for two-step consecutive binding with rate constants of 7.72 mM-1 min-1 and 0.088 min-1. The resulting PLP-HSA complex had absorption peaks at 338 and 414 nm and was reduced by potassium borohydride. The 414-nm peak is probably due to a protonated aldimine formed between PLP and HSA. The binding of PLP to bovine serum albumin (BSA) at equimolar concentrations at pH 7.4 occurred at about 10% the rate of its binding to HSA. The final PLP-BSA complex absorbed maximally at 334 nm and did not appear to be reduced with borohydride. Equilibrium dialysis of PLP and HSA indicated that there were more than one class of binding sites of HSA for PLP. There was one high affinity site with a dissociation constant of 8.7 microM and two or more other sites with dissociation constants of 90 microM or greater. PLP binding to HSA was inhibited by pyridoxal and 4-pyridoxic acid. It was not inhibited appreciably by inorganic phosphate or phosphorylated compounds. The binding of PLP to BSA was inhibited more than its binding to HSA by several compounds containing anionic groups. It is concluded that PLP binds differently to HSA than it does to BSA.
Fonda
M L
ML
Department of Biochemistry, University of Louisville School of Medicine, Kentucky 40292.
Trauss
C
C
Guempel
U M
UM
D001665:Binding Sites; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000097:blood / Q000378:metabolism*; D011736:Pyridoxine / Q000031:analogs & derivatives / Q000494:pharmacology; D012709:Serum Albumin / Q000378:metabolism*; D013053:Spectrophotometry
D012709:Serum Albumin; D011732:Pyridoxal Phosphate; D011736:Pyridoxine