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 (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 alpha-helical content of BSA and no change in the beta-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 (>10(6) M-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.
Zhang
F
F
Department of Biomedical Sciences, University at Albany, State University of New York, USA.
Thottananiyil
M
M
Martin
D L
DL
Chen
C H
CH
D000595:Amino Acid Sequence; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002942:Circular Dichroism; D004789:Enzyme Activation; D005454:Fluorescence Polarization; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D012709:Serum Albumin; D011732:Pyridoxal Phosphate; D005968:Glutamate Decarboxylase