Carbohydrate-linked asparagine-101 of prothrombin contains a metal ion protected acetylation site. Acetylation of this site causes loss of metal ion induced protein fluorescence change.
Prothrombin fragment 1 (prothrombin residues 1-156) contains two acetylation sites that are protected from derivatization by calcium. The first site was protected by only calcium [Welsch, D. J., & Nelsestuen, G. L. (1988) Biochemistry (second of three papers in this issue)] while the second site was protected by magnesium as well. To identify this second acetylation site, fragment 1 was first acetylated with unlabeled reagent in the presence of magnesium. Metal ions were removed, and the protein was acetylated with radiolabeled reagent. The incorporated radiolabel was stable over long periods of time and at acidic or basic pH as long as elevated temperatures were avoided. The radiolabel was removed by treatment of the protein at pH 10 and 50 degrees C or with 0.2 M hydroxylamine at 50 degrees C for at least 30 min. Proteolytic degradation of the protein showed that the radioactivity appeared in a tryptic peptide corresponding to residues 94-111 of prothrombin. The Lys-97 in this peptide was acetylated but did not contain radiolabel. Amino acid sequence analysis revealed that the radiolabel was associated with an unextracted sequence product. Aglycofragment 1, produced by treatment of fragment 1 with HF, was radiolabeled by this procedure; peptide 94-111 was isolated and was further digested with protease. The major radiolabeled product contained Asn101-Ser102 along with the expected chitobiose attached to Asn-101. NMR analysis revealed the presence of three acetate groups which would correspond to two from the chitobiose plus the incorporated acetate residue.(ABSTRACT TRUNCATED AT 250 WORDS)
Welsch
D J
DJ
Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul 55108.
Nelsestuen
G L
GL
D000107:Acetylation; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001216:Asparagine; D001665:Binding Sites; D002417:Cattle; D004220:Disulfides; D007460:Iodoacetamide / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014357:Trypsin
D000596:Amino Acids; D004220:Disulfides; D010446:Peptide Fragments; D011498:Protein Precursors; D001216:Asparagine; C029733:prothrombin fragment 1; D011516:Prothrombin; D014357:Trypsin; D008274:Magnesium; D007460:Iodoacetamide