Chemical modification of prothrombin fragment 1: documentation of sequential, two-stage loss of protein function.
The amino groups of prothrombin fragment 1 (amino acids 1-156 of prothrombin) were derivatized by acetylation, amidination, and reductive methylation. Conditions that caused complete acetylation of protein amino groups produced a fragment 1 derivative which no longer displayed a metal ion dependent intrinsic fluorescence change and had lost its membrane binding capability as well. However, when derivatized in the presence of calcium ions, extensive acetylation yielded a product that underwent protein fluorescence quenching at metal ion concentrations similar to those observed for the native protein. This derivative bound to membranes in a calcium-dependent manner with only a small reduction in affinity. Several results showed the existence of a partially functional protein that was characterized by a high degree of calcium-dependent protein fluorescence quenching but which had a requirement for 10-fold higher calcium concentration. This derivative was produced by partial acetylation (greater than 3 equiv) of metal-free protein. This partially acetylated protein had greatly diminished membrane binding. The calcium-protected amino group, therefore, was among the most reactive acetylation sites in the metal-free protein. The second site, responsible for abolishing all metal ion induced fluorescence change, was resistant to acetylation and became derivatized at the last stages of amino group acetylation.(ABSTRACT TRUNCATED AT 250 WORDS)
Welsch
D J
DJ
Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul 55108.
Pletcher
C H
CH
Nelsestuen
G L
GL
D000107:Acetylation; D000578:Amidines; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008745:Methylation; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000578:Amidines; D010446:Peptide Fragments; D011498:Protein Precursors; C029733:prothrombin fragment 1; D011516:Prothrombin; D002118:Calcium