Structural and functional relationships in 5'-nucleotidase from bull seminal plasma. A Fourier transform infrared study.
Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structure of 5'-nucleotidase from bull seminal plasma (BSP). Spectra of protein in both D2O and H2O were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide I band. The protein, which is made up of two apparently identical subunits and which contains two zinc atoms, was studied in its native form, in the presence of dithiotreitol (DTT) and after removal of the two zinc atoms by means of nitrilotriacetic acid (NTA). Deconvolved and second derivative spectra of amide I band showed that the native protein contains mostly beta-sheet structure with a minor content of alpha-helix. The quantitative analysis of the amide I components was performed by a curve-fitting procedure which revealed 54% beta-sheet, 18% alpha-helix, 22% beta-turns and 6% unordered structure. The second derivative and deconvolved spectra of amide I band showed that no remarkable changes in the secondary structure of 5'-nucleotidase were induced by either DTT or NTA. These results were confirmed by the curve-fitting analysis where little or no changes occurred in the relative content of amide I components when the protein was treated with DTT or with NTA. Major changes, however, were observed in the thermal denaturation behavior of the protein. The native protein showed denaturation at temperatures between 70 and 75 degrees C, while the maximum of denaturation was observed between 65 and 70 degrees C and between 55 and 60 degrees C in the presence of NTA and DTT, respectively. The results obtained indicate that the two separate subunits of the protein have essentially the same secondary structure as that of the native enzyme.
Fini
C
C
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Perugia, Italy.
Bertoli
E
E
Albertini
G
G
Floridi
A
A
Tanfani
F
F
D015720:5'-Nucleotidase / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004229:Dithiothreitol / Q000378:metabolism; D005583:Fourier Analysis; D008297:Male; D009571:Nitrilotriacetic Acid / Q000378:metabolism; D011487:Protein Conformation; D012661:Semen / Q000201:enzymology*; D013055:Spectrophotometry, Infrared
D015720:5'-Nucleotidase; D009571:Nitrilotriacetic Acid; D004229:Dithiothreitol