Heparitinase digestion. Part 2
The hbs-wt, hbs-kara, an assortment of V3 loop peptides, and RANTES were bound to a heparin-Sepharose column and eluted with 0.01, 0.15, 0.5, 1.0 or 2.0 M NaCl. The NaCl concentration required to elute the peptides provides a relative value for the affinity between the peptide and heparin, and is directly proportional to the Kd value. The 0.15 M NaCl concentration is physiologically relevant but reflects weak binding, 0.5 M indicates moderate binding, and 1.0 M and above represents strong binding (reviewed in chapter 6 of “Heparin-Binding Proteins”. Most of the peptides could be detected in the fractions by their absorbance of light at 280 nm on a spectrophotometer, with the exception of the linear V3 loop peptide of strain IIIB that lacks aromatic side chains. The Pierce BCA Protein Assay was used to detect this peptide.
The hbs-wt peptide eluted at 0.5 M NaCl (Table 1). The hbs-kara peptide did not bind to the column at all, eluting with the wash buffer. In a subsequent experiment, without the wash step, it eluted with 0.01 M NaCl. The cyclic V3 loop peptide of X4 HIV strain MN also eluted at 0.5 M as did the linear peptide of X4 strain IIIB. Of the linear peptides that are overlapping subunits of the MN cyclic peptide, the peptide containing the consensus heparin binding motif eluted at 1.0 M. This is a stronger interaction than the cyclic full-length V3 peptide. Other V3 peptides based on consensus sequences of subtypes B and EA, or specific strains RF and SF2, eluted at 0.15 M. RANTES eluted at 0.5 M. There was no direct relationship between net charge of the peptides and affinity to heparin, as the cyclic MN peptide with a charge of +7 eluted at a lower NaCl concentration than the linear peptide at a charge of +6. The most neutral net charge in the tested group of peptides was 0 for the hbs-wt, and it eluted at 0.5 M NaCl.
An ELISA based on coating plates with BSA-heparin and determining the binding of the hbs-wt peptide, or hbs-kara as a control, was developed. This assay showed high sensitivity for detecting bound hbs-wt peptide with low background as demonstrated by low signal produced by the hbs-kara control (data not shown). The binding was inhibited at the same NaCl concentrations that eluted the hbs-wt peptide in the heparin-Sepharose column at 0.5 M and above (data not shown). When sulfated polysaccharides were added to the ELISA, they inhibited the binding of the hbs-wt peptide in a dose-dependent manner. The same sulfated polysaccharides found to be inhibitory in the erythrocyte invasion assay and the PvRII region binding assay where inhibitory in the ELISA, with no inhibition from chondroitin sulfate C.