Abstract
Abstract Part Ⅰ What is the possible role of the conformation of heparin-binding peptide in its interaction with heparin? This question that was undertaken solved in this project. To achieve this, ApaK6 (O) (NH2-CNCKAPETAKCAKQCQKAQKAQAKQAKQW-NH2) with 2 cysteine bridges was designed. In water, ApaK6 (O) adopt α-helical conformation, and its disulfide reduced form (ApaK6 (R)) was random coil conformation. Initially filtration was done with heparin oligosaccharide. Both ApaK6 (O) and ApaK6 (R) could be induced to formα-helical conformation by heparin oligosaccharide. However heparin is highly inhomogeneous. For detailed analysis, this form of heparin was enzymatically digested and extensively purified to obtain homogenous oligosaccharide fragments. For matching the binding size, the heparin octasaccharide Ⅷ was selected for assay. It was found that the octasaccharide Ⅷ fragment with the highest possible sulfation was relatively aboundant, among the products obtained from the enzymatic digestion.By using octasaccharide Ⅷ for assaying its interaction with ApaK6 (O) and Apak6 (R), the following could be inferred: 1. The dissociation constant of ApaK6 (O)-octasaccharide Ⅷ (202.5 μM) was less than ApaK6 (R)-octasaccharide Ⅷ (497.8 μM) at physiological condition. 2. Upon interaction with octasaccharide Ⅷ, the stoichiometry of their binding was different. Octasaccharide Ⅷ provided two binding sites for ApaK6 (O), but provided only one binding site for ApaK6 (R). This could be due to compact geometry of ApaK6 (O), owing to the presence of two disulfide bridges compared disulfide bridge deficient ApaK6 (R). 3. From the plot of ionic strength dependence of octasachharide Ⅷ on ApaK6 (O) and ApaK6 (R) interaction, the slopes indicate that ~4 and ~5 ionic interactions contribute to both ApaK6 (O) and ApaK6 (R) interactions with octasaccharide Ⅷ. Again the intercepts indicated an different nonionic contribution between their interactions with octasaccharide Ⅷ. The DGnonionic contribution of ApaK6 (O)- octasaccharide Ⅷ interaction was -10.2 KJ/mol, and one of ApaK6 (R) was -4.8 KJ/mol. The nonionic contribution contains hydrogen bonding, hydrophobic effect, and van der Waal interaction. Thus, from above results, it seemed that the conformation of the peptide. It was believed why the heparin-binding domains of heparin-binding proteins adopt such conformations was reasonable. 4. Upon surveying ApaK6 (O) and ApaK6 (R) interaction with different sulfation density of heparin octasaccharide, it was found that the most sulfation did not necessarily result in the largest association constant. It might be the sulfation density of heparin octasccharide was not the only factor for determining the binding strength with the peptides. PartⅡ The binding of glycosaminoglycans to a synthetic peptide (SKAQKAQAKQAKQAQKAQKAQAKQAKQW-CONH2), K828, consisting of a hybrid consensus heparin binding sequence was studied using circular dichroism and fluorescence anisotropy. The result showed among all GAGs, only those containing more iduronic acid (IdoA)- containing GAGs, like DS, HS, and LMWH, could transfer helical conformation in K828. The increasing IdoA residues in the GAGs is in the order of LMWH > HS > DS. The more IdoA residues, the GAG contained, the more contents of helical structure K828 formed. The strength of their interactions was also evaluated by thermal denaturation of K828, upon complexing with DS, HS, and LMWH. There seemed to be a good correlation between the percentage of helix induced in the peptide backbone and the stability of K828. The more IdoA residues, the GAG contained, the more strength interactions K828 involved in.