Conformational alteration in serum albumin as a carrier for pyridoxal phosphate: A distinction from pyridoxal phosphate-dependent glutamate decarboxylase

The conformation of bovine serum albumin (BSA), a pyridoxal phosphate (pyri doxal-P) carrier, was investigated by using uv/visible spectrophotometry, f luorescence spectroscopy, circular dichroism, and differential scanning mic rocalorimetry, 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 (exc ited at 295 or 280 nm) by 24% and enhances fluorescence steady-state polari zation of BSA by 20%. These observations suggest a conformational change in BSA when it interacts with pyridoxal-P. However, this conformational chang e appears to be small since circular dichroism showed only a 2-4% decrease in the cu-helical content of BSA and no change in the beta-sheet content, a nd differential scanning microcalorimetry yielded only a 10% change in the enthalpy of thermal unfolding of BSA. 2-Aminoethylisothiouronium bromide, a n antioxidant, causes no effect on either uv/visible absorption spectrum or fluorescence emission intensity of BSA, suggesting that BSA lacks sensitiv e sulfhydryl groups. To help in understanding BSA as a carrier for pyridoxa l-P, the results were compared with those for glutamate decarboxylase (GAD) , a pyridoxal-P-dependent protein, which requires pyridoxal-P as the cofact or for activity, Although BSA and GAD exhibit comparable molecular weights (66430 versus 65300), numbers of amino acid residues (582 versus 585), and binding affinity (>10(6) M-1), distinct conformational alterations occur be tween the two proteins upon interacting with pyridoxal-P: a small conformat ional change for BSA versus a large conformational change for GAD, In contr ast to the case of BSA, AET causes significant effects on both the uv/visib le spectrum and fluorescence emission intensity of GAD, because GAD contain s sensitive sulfhydryl groups. Factors such as disulfide bond and active si te sequence were discussed to understand BAS as a carrier for pyridoxal-P a nd a pyridoxal-P-independent protein. (C) 1999 Academic Press.