HEPARIN BINDING TO HUMAN PLASMA LOW-DENSITY LIPOPROTEINS - DEPENDENCE ON HEPARIN SULFATION DEGREE AND CHAIN-LENGTH

Binding between low-density lipoproteins (LDL) and fluorescein-labeled heparin was studied quantitatively with a modified form of a published procedure [Cardin, A. D., Randall, C. I., Hirose, N., & Jackson, R. L. (1987) Biochemistry 26, 5513-5518], using fluorescence anisotropy titrations. Assumption of binding site equivalence satisfactorily interpreted experimental data. Accordingly, the apparent total capacity, n, and the average dissociation constant, K(d), were estimated as n almost-equal-to 24 disaccharides per LDL particle and K(d) almost-equal-to 4-mu-M in 0.05 M HEPES/0.1 M NaCl, pH 7.4, 22-degrees-C. Competition experiments with unlabeled heparins were exploited for the quantitative study of K(d) as a function of heparin chain length and sulfation degree (ns = sulfate groups per disaccharide). The former parameter was investigated with a series of bovine lung heparin fractions with M(w) ranging from 1800 to 21 000 and constant sulfation degree (n(s) = 2.8 +/- 0.1). A series of physically fractionated or chemically modified heparins having 1.2 < n(s) < 3.5 were used to explore the dependence on sulfation degree. LDL affinity was found to increase with increasing both n(s) and M(w): an empirical M(w)-1.6 dependence represented very well the chain length data set; a linear dependence was observed for log K(d) as a function of n(s), after appropriate allowance was made for chain length differences among samples. This regularity confirmed that LDL-heparin binding is mainly driven by electrostatic forces. Consistently, the K(d) dependence on heparin chain length was analyzed in terms of a linear lattice binding model requiring about 10 disaccharides to fit each independent binding site on apolipoprotein B-100, the protein component of each LDL particle. Furthermore, this model implies an approximately 2-fold increase of LDL binding capacity with respect to the estimate of the equivalent site model, as a consequence of a statistical site exclusion effect. When this correction was applied, each LDL particle was found to contain 4-5 heparin binding sites, each capable of binding about 10 disaccharidic units. Together with the steep dependence of the dissociation constant on salt concentration (d log K(d)/d [NaCl] = 8.2 +/- 0.3), this result indicates that about 40-50 positively charged residues in apoB-100 are involved in heparin binding.