Table 2.

Quantities of identifiable guanine nucleotides found in standard and mutant forms of HRas as determined by percentage peak area and concentration.

HRas	GMP (%)	GMPPN (%)	GDP (%)	GMPPNP (%)	GTP (%)	Nucleotides arising from active HRas (%)	Nucleotides arising from inactive HRas (%)	Range of Average [Total Nucleotide], (μM)	Range of Average [Total Protein], (μM) & (Mole Ratio of Nucleotide : Protein)
HRas¥ (2 X 3)	0.8 ± 0.7	75 ± 2	18.5 ± 0.5	3 ± 1	2.5 ± 0.7	81 ± 4	19 ± 1	48.1–48.2	N/A
G12S¥ (2 X 3)	8 ± 2	70 ± 2	9 ± 2	0.4 ± 0.5	14.4 ± 0.3	83 ± 3	17 ± 4	39.6–40.2	N/A
E37G¥ (2 X 3)	Not measurable	87.3 ± 0.5	7.4 ± 0.8	3 ± 1	2 ± 1	93 ± 3	7.4 ± 0.8	40.3–47.8	N/A
D38E¥ (2 X 3)	1.7 ± 0.4	67.8 ± 0.7	27.4 ± 0.9	2.9 ± 0.2	0.3 ± 0.6	71 ± 2	29 ± 1	33.2–37.7	N/A
E63K¥ (2 X 3)	1.3 ± 0.7	67 ± 1	16.5 ± 0.5	3 ± 1	11.9 ± 0.6	82 ± 3	18 ± 1	33.2–37.7	N/A
Y64G¥ (2 X 3)	0.7 ± 0.4	67.8 ± 0.8	14.9 ± 0.2	2.5 ± 0.8	14.1 ± 0.2	84 ± 2	15.6 ± 0.6	49.4–57.1	N/A
HRas (6 X 3) *	2.8 ± 0.8	74 ± 2	19.0 ± 0.8	2.1 ± 0.7	2.8 ± 0.8	78 ± 3	22 ± 2	27.2–42.1	29.4–45.6 (1:1.04 ± 0.03)
HRas GDP* (3 X 3)	3.9 ± 0.7	N/A	96.1 ± 0.7	N/A	Not measurable	Not measurable	100 ± 1	21.3–41.8	20.0–40.1 (1:0.95 ± 0.01)

^¥Samples were generated using wash protocol A. A total of two samples obtained from a single protein preparation were applied to the HPLC system, and measurements recorded in triplicate.

^*Samples were generated using wash protocol B. In this case, a total of three samples from a single protein preparation (HRas GDP) or six samples (HRas) obtained from two separate protein preparations were applied to the HPLC system, and measurements recorded in triplicate. All values for individual samples are reported in Table S1. (Further studies of G12S are needed to ascertain whether the unusually high level of GMP observed for this mutant is reproducible over multiple protein preparations, and whether it may reveal unusual hydrolytic features of the mutant active site).