TABLE 1.

Binding of heparin to diverse CPCs

	ITC							DLS
Ligand and charge (zCPC) at given pH*	Kd,i (nM)†	n binding sites per heparin‡ (nligands/nheparin)	n × zCPC	Kd,1 (nM)§	Reaction enthalpy (kcal/mol CPC)	Entropy (kcal /mol CPC)	Heat capacity change¶ (cal/mol CPC/K)	Rh** (nm)	FWHM†† of Rh (nm)
HIV Tat-PTD8+	443 ± 178	6.68 ± 0.57	53.4 ± 4.55	67.9 ± 30.5	−12.3 ± 0.33	−3.47 ± 0.37	38.6	94.4 ± 5.9	36.1 ± 6.6
Penetratin7+	338 ± 105	6.79 ± 0.45	47.5 ± 3.18	50.6 ± 19.0	−11.6 ± 0.12	−2.63 ± 0.18	−128	129 ± 18.7	51.1 ± 8.4
Nona-l-arginine9+	459 ± 115	5.38 ± 0.62	48.4 ± 5.54	87.9 ± 33.3	−12.8 ± 2.65	−4.02 ± 2.80	89.3	98.3 ± 6.2	36.4 ± 5.2
PLL1616+	543 ± 253	2.91 ± 0.30	46.6 ± 4.86	198 ± 108	−8.50 ± 0.11	0.18 ± 0.30	11.0	77.7 ± 6.8	24.8 ± 6.9
LPEI≈20+ (2.5 kDa); pH 7.4	741 ± 104	3.36 ± 0.51	67.3 ± 10.2	224 ± 52.1	−16.5 ± 2.12	−8.08 ± 2.20	−235	131 ± 14.1	28.9 ± 7.2
LPEI≈58+ (2.5 kDa); pH 5.0	531 ± 221	1.10 ± 0.27	64.0 ± 15.9	467 ± 94.9	−37.1 ± 4.98	−28.4 ± 4.1	−76.1	123 ± 12.2	38.1 ± 4.1
DOTAP1+ (as 30-nm small unilamellar vesicles; nDOTAP: nDOPC = 3:7)	1345 ± 417	31.5 ± 3.09	31.5 ± 3.09	45.7 ± 19.0	−0.49 ± 0.07	7.82 ± 0.53	−25.7	586 ± 149	428 ± 136
Acridine orange1+	868 ± 293	65.7 ± 6.8	65.7 ± 6.8	13.3 ± 4.6	−3.82 ± 0.27	4.59 ± 0.48	−32.0	311 ± 56.7	234 ± 92.5
Ethidium bromide1+	No binding observed‡‡							<10
Propidium iodide2+	No binding observed							<10
l-arginine1+	No binding observed							<10

^*Conditions and pH (7.4) as described in Fig. 2; LPEI was measured additionally at pH 5.00 (133 mM NaCl, 30 mM acetate). Results are reported as mean ± SD from four individual sample preparations.

^†Microscopic dissociation constant (K_d,i) of n individual binding sites found per heparin chain.

^‡Stoichiometry as experimentally determined (ITC) for 100% of heparin's multiple binding sites saturated with the specific ligand; heparin had a sulfur content of 11.3%, yielding an average of 45.8 sulfate groups or a total of 68.2 negative charges (including carboxyl groups) per heparin (average mol wt 13,000).

^§Macroscopic dissociation constant (K_d,1) for the first accessible binding site in the GAG.

^¶Molar heat capacity change at constant pressure () as calculated from the slope of as a function of different temperatures (8, 18, 28, 38, 48, and 58°C).

^**Hydrodynamic radius (R_h) as experimentally determined for 50% of heparin's binding sites (13.7 μM, 1.4 ml) saturated with the ligand; except for AOR and DOTAP, which were measured at 10% and 5% saturation of heparin, respectively, because at higher molar ratios, R_h exceeded the laser's wavelength. At current settings, the lower detection limit was ∼10 nm.

^††Width of the R_h distribution was calculated as full width at half-maximum (FWHM).

^‡‡Physiological ionic strength apparently prevents binding of ethidium bromide to heparin (32).