Conformational Alteration in Serum Albumin as a Carrier for Pyridoxal Phosphate: A Distinction from Pyridoxal Phosphate-Dependent Glutamate Decarboxylase

Abstract

The conformation of bovine serum albumin (BSA), a pyridoxal phosphate (pyridoxal-P) carrier, was investigated by using uv/visible spectrophotometry, fluorescence spectroscopy, circular dichroism, and differential scanning microcalorimetry. Upon interacting with pyridoxal-P, the uv/visible absorption spectrum of BSA exhibits peaks at 330 and 392 nm due to the formation of a Schiff base. Pyridoxal-P quenches the fluorescence emission intensity (excited at 295 or 280 nm) by 24% and enhances fluorescence steady-state polarization of BSA by 20%. These observations suggest a conformational change in BSA when it interacts with pyridoxal-P. However, this conformational change appears to be small since circular dichroism showed only a 2–4% decrease in the α-helical content of BSA and no change in the β-sheet content, and differential scanning microcalorimetry yielded only a 10% change in the enthalpy of thermal unfolding of BSA. 2-Aminoethylisothiouronium bromide, an antioxidant, causes no effect on either uv/visible absorption spectrum or fluorescence emission intensity of BSA, suggesting that BSA lacks sensitive sulfhydryl groups. To help in understanding BSA as a carrier for pyridoxal-P, the results were compared with those for glutamate decarboxylase (GAD), a pyridoxal-P-dependent protein, which requires pyridoxal-P as the cofactor for activity. Although BSA and GAD exhibit comparable molecular weights (66430 versus 65300), numbers of amino acid residues (582 versus 585), and binding affinity (>106M−1), distinct conformational alterations occur between the two proteins upon interacting with pyridoxal-P: a small conformational change for BSA versus a large conformational change for GAD. In contrast to the case of BSA, AET causes significant effects on both the uv/visible spectrum and fluorescence emission intensity of GAD, because GAD contains sensitive sulfhydryl groups. Factors such as disulfide bond and active site sequence were discussed to understand BAS as a carrier for pyridoxal-P and a pyridoxal-P-independent protein.