Table 4. Assessment of the Amounts of... | American Society of Hematology

Table 4.

Assessment of the Amounts of Gla and Glu Residues at Each of the Potential γ-Carboxylation Sites in the Gla Domain of Protein C From the Patient

Cycle	Gla(Glu)-residue		Fraction 30 [mutant:normal (3:1)]			Fraction 31 [mutant:normal (1:2)]
	Mutant	Normal	Glu	Gla	Relative amount of Gla (%)	Glu	Gla	Relative amount of Gla (%)
			(Δ mAU)	(Δ mAU)		(Δ mAU)	(Δ mAU)
6	—	6	0.00	1.10	100	0.06	2.21	97
7	6	7	0.07	4.33	98	0.13	2.74	95
8	7	—	0.11	3.66	97	0.08	1.06	93
14	—	14	0.02	0.29	94	0.08	0.63	89
15	14	—	0.05	1.25	96	0.06	0.53	90
16	—	16	0.01	0.62	98	0.04	0.72	95
17	16	—	0.09	1.51	94	0.11	0.77	88
19	—	19	0.00	0.43	100	0.07	0.67	91
20	19	20	0.09	1.25	93	0.07	0.84	92
21	20	—	0.05	1.30	96	0.04	0.50	93
25	—	25	−0.01	0.10	100	0.03	0.24	89
26	25	26	0.06	0.43	88	0.11	0.53	83
27	26	—	0.05	0.96	95	0.04	0.34	89
29	—	29	0.01	0.34	97	0.03	0.36	92
30	29	—	0.06	1.01	94	0.03	0.24	89

Cycle	Gla(Glu)-residue		Fraction 30 [mutant:normal (3:1)]			Fraction 31 [mutant:normal (1:2)]
	Mutant	Normal	Glu	Gla	Relative amount of Gla (%)	Glu	Gla	Relative amount of Gla (%)
			(Δ mAU)	(Δ mAU)		(Δ mAU)	(Δ mAU)
6	—	6	0.00	1.10	100	0.06	2.21	97
7	6	7	0.07	4.33	98	0.13	2.74	95
8	7	—	0.11	3.66	97	0.08	1.06	93
14	—	14	0.02	0.29	94	0.08	0.63	89
15	14	—	0.05	1.25	96	0.06	0.53	90
16	—	16	0.01	0.62	98	0.04	0.72	95
17	16	—	0.09	1.51	94	0.11	0.77	88
19	—	19	0.00	0.43	100	0.07	0.67	91
20	19	20	0.09	1.25	93	0.07	0.84	92
21	20	—	0.05	1.30	96	0.04	0.50	93
25	—	25	−0.01	0.10	100	0.03	0.24	89
26	25	26	0.06	0.43	88	0.11	0.53	83
27	26	—	0.05	0.96	95	0.04	0.34	89
29	—	29	0.01	0.34	97	0.03	0.36	92
30	29	—	0.06	1.01	94	0.03	0.24	89

Fractions 30 and 31 (see Fig 3) were methylated and subjected to N-terminal sequence analysis. The ratio between the mutant and normal N-terminal sequence was 3:1 in fraction 30 and 1:2 in fraction 31, as estimated from the yields of amino acids in the first six cycles (compare with Table 2, where equal amounts of the two forms were determined). Methylation of the samples allowed the positive identification of Gla residues (see Fig 5). The amounts of Gla and Glu at each potential γ-carboxylation site are expressed as the change in absorbance at 269 nm of methylated PTH-Gla and PTH-Glu derivatives. Assuming that the two PTH derivatives have comparable extinction coefficients, the relative amounts of Gla were calculated as Δ mAU_Gla/(Δ mAU_Gla + Δ mAU_Glu). For further details, see Materials and Methods. The relative amount of Gla [median (range)] was 96% (88-100%) in fraction 30 and 91% (83-97%) in fraction 31. It is concluded from the data that there is no significant difference in the relative Gla content of the mutant and normal Gla-domains.

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