Vitamin K-dependent carboxylase: minimized escape of CO2 from solution may prolong linearity of the reaction rate

Escape of 14CO2 from the reaction mixture into the gas phase may seriously affect the accuracy of in vitro measurement of vitamin K-dependent carboxylase activity (and probably that of other carboxylases as well). In this paper we describe the effect of (a) the volume of the test tubes in which the reaction is performed, (b) the addition of an excess of NaH12CO3 in parallel with standard amounts of NaH14CO3, and (c) the incubation temperature. In this way optimal conditions are defined and used for the carboxylation of various peptide and protein substrates. It is shown that both a prosequence and an internal recognition site contribute to the effective recognition of a substrate by carboxylase. The maximal efficiency of carboxylation was 1-2% with substrates lacking both signals and 20-50% if only one was present. This indicates the need for developing peptide substrates containing both recognition signals for vitamin K-dependent carboxylase.