. 2003 May;12(5):903–913. doi: 10.1110/ps.0235703

Table 1.

Summary of experimental results

						Thermally induced unfolding	Urea-induced unfolding
Solution conditions	Aggregation observed	Refractive index increment dn/dc (mL/g)	Mass averaged molecular weight (kD)	B₂₂ × 10³ (cm³mole/g²)	B₂₂/B₂₂^{HS^b}	Apparent T_m (M)	C_m (M)	m (kcal/mole/M)	ΔG_unf (25°C) (kcal/mole)
pH 3.5 HCl	No	0.187	18.5 ± 3.1	13.5 ± 3.3	85.5 ± 21	74.3 ± 1.7	5.15 ± 0.11	2.19 ± 0.14	11.3 ± 0.71
pH 3.5 HCl 150 mM NaCl	Yes	0.186	N.D.^d	N.D.^d	N.D.^d	64.3 ± 0.19	5.59 ± 0.26	1.77 ± 0.16	9.33 ± 0.63
pH 3.5 HCl 0.26 M Sucrose	No	0.173	30.4 ± 3.0	19.8 ± 1.0	125 ± 6.5	75.4 ± 0.35
pH 3.5 HCl 0.50 M Sucrose	No	0.160	56.5 ± 23	20.9 ± 3.5	264 ± 44^c	77.1 ± 0.43
pH 3.5 50 mM NaAc	No	0.186	15.6 ± 1.4	8.98 ± 1.8	56.9 ± 11	75.1 ± 0.49
pH 3.5 100 mM NaAc	No	0.186	16.4 ± 0.70	6.88 ± 0.75	43.6 ± 4.8	74.2 ± 0.92
pH 3.5 150 mM NaAc	No	0.186	17.8 ± 1.6	4.25 ± 1.1	26.9 ± 6.9	72.5 ± 0.41	5.37 ± 0.15	1.96 ± 0.12	10.52 ± 0.40
pH 3.5 200 mM NaAc	No	0.186	19.0 ± 0.56	2.51 ± 0.32	15.9 ± 2.0	73.6 ± 0.96
pH 3.5 200 mM NaAc 0.50 M Sucrose	No	0.163	22.5 ± 1.7	3.39 ± 0.67	21.5 ± 4.2	73.9 ± 0.51
pH 6.1 PBS	Yes	0.190	16.8 ± 0.99	−1.33 ± 0.97	−8.42 ± 6.2	63.4 ± 0.57	5.18 ± 0.13	2.21 ± 0.07	11.4 ± 0.61
pH 6 10 mM Sodium Citrate	Yes	0.196	20.0 ± 0.74	−1.86 ± 0.68	−11.8 ± 4.3
pH 7 PBS	Yes (7.3 ± 0.6)^a	0.189	19.7 ± 0.57	−2.34 ± 1.9	−14.8 ± 12	61.1 ± 2.0^a	5.88 ± 0.22	1.61 ± 0.05	9.48 ± 0.49
pH 7 10 mM Sodium Phosphate	Yes	0.190	23.9 ± 0.92	−0.72 ± 0.45	−4.57 ± 2.8
pH 7 PBS 0.050 M Sucrose	Yes	0.176	17.9 ± 0.35	−1.52 ± 0.45	−9.63 ± 2.9	60.8 ± 0.49
pH 7 PBS 0.15 M Sucrose	Yes	0.174	18.1 ± 0.45	−1.20 ± 0.33	−7.60 ± 2.1	60.6 ± 1.4
pH 7 PBS 0.25 M Sucrose	Yes (4.8 ± 1.7)^a	0.176	33.0 ± 6.7	16.3 ± 3.9	206 ± 49^c	63.0 ± 1.3^a
pH 7 PBS 0.50 M Sucrose	Yes (2.8 ± 1.5)^a	0.179	30.1 ± 14	16.9 ± 1.8	214 ± 24^c	63.7 ± 2.0^a
pH 7 PBS 0.75 M Sucrose	Yes (1.4 ± 1.3)^a	0.163	33.2 ± 11	9.30 ± 7.4	118 ± 94^c	65.5 ± 2.1^a
pH 7 PBS 1.0 M Sucrose	Yes (0.7 ± 0.2)^a	0.155	33.4 ± 11	17.4 ± 4.3	220 ± 55^c	68.2 ± 1.8^a

^a Results from Krishnan et al. (2002). Numbers in parenthesis of aggregation observed in pH 7 PBS are initial aggregation rates with units μmole/L/day.

^bB₂₂^HS was calculated as four times the molecular volume (V) of rhGCSF using a molecular weight (M) of 19.6 kD for monomers and 39.2 kD for dimers and a protein specific volume (ν) of 0.75 cm³/g (B₂₂^HS = 4V/M² = 4 ν/M).

^cB₂₂ normalized using dimer B₂₂^HS; other calculations were carried out using monomer B₂₂^HS. In pH 7 PBS solutions with 0.25 M and higher sucrose concentrations, extrapolated molecular weights correspond to 40–60 mole % dimer.

^d Could not be reliably determined due to precipitation during sample preparation (dialysis at 4°C) for light scattering experiment.

Errors associated with extrapolated molecular weights and B₂₂ values are standard errors from the linear regression of light scattering data using equation (2). Errors associated with unfolding experiments results are standarddeviations from triplicate samples.