Skip to main content
. 1999 Jun 8;96(12):7065–7070. doi: 10.1073/pnas.96.12.7065

Figure 2.

Figure 2

HPLC and fluorescence measurements of the hydrolysis reaction of Ras-proteins loaded with either GTP or GTP-analogues. (a) Single turnover hydrolysis of 150 μM wild-type Ras (closed symbols) and A61Ras (open symbols) with 140 μM of either GTP (triangles) or DABP-GTP (circles) was measured in standard buffer at 30°C. Aliquots of the reaction mixture at the indicated time points were analyzed by HPLC as described in Materials and Methods. (b) HPLC analysis of DABP-GTPase reaction products by wild-type Ras after 0, 1, 5, and 30 min. Elution times of standards GDP (3.7 min), DABP-GTP (5.7 min), and DABP-Pi (7.3 min) are indicated. (c) Real time tryptophane fluorescence kinetics of the hydrolysis of GTP and various GTP-derivatives illustrated in Fig. 1 were followed by rapid-mixing of 1.1 μM nucleotide-free W32Ras with 1 μM of the respective GTP-analogues in standard buffer at 30°C by using stopped-flow apparatus. For calculation of the rate constants, the data were fitted to a single exponential.