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 (>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. The second site did not function as a substitute for the first site. That is, both sites were shown to be essential for full protein function so that calcium actually protected both sites from acetylation. The second site, but not the first site, could undergo deacetylation under alkaline conditions and mild heat (pH 10, 50°C) or with hydroxylamine and mild heat (0.2 M, 50°C). Thus, the fully acetylated protein could be returned to its intermediate state of function (high calcium requirement, low membrane binding) by these treatments. Amidination and reductive methylation of fragment 1 produced derivatives which were similar to the partially acetylated protein. These derivatives underwent protein fluorescence quenching which required 10-fold more calcium than native protein and had greatly reduced membrane affinity. This indicated that these subtle changes abolished the function of the first site but did not alter the function of the second site.