Abstract
Abstract： Glycosaminoglycans (GAGs) have the highest negative charge density in extracellular matrix of all tissues and perform a myriad of physiological functions. Glycosaminoglycans exist as proteoglycans, and these barriers in extracellular matrix played important roles on regulated the function of GAGs associated proteins. (Sawitzky, D., 1996) The compositions of GAG chains of the proteoglycans are distinct and vary in different tissues. (Jackson, et al., 1991) The previous research of the association between GAGs and proteins focused on the cell-cell adhesion, signal transduction, etc. (Templeton, D. M., 1992) Heparin and heparan sulfate belong to the glucosaminoglycan subclass of GAGs and attract special attention because they exhibit greatest structural diversity. (Gallagher, et al., 1992) Numerous proteins, such as fibroblast growth factors, (Faham, et al., 1996) antithrombin III, (Petitou, et al., 1988) phospholipase A2, (Sartipy, et al., 1996; Lomonte, et al., 1994) among others, bind to heparin. Our lab recently found that cardiotoxins of snake venom, with selective cytolytic activity, also bind to heparin and heparan sulfate under physiological conditions. Cobra venom cardiotoxins are a family of highly homologous, consisting of 60 amino acid residues, and basic polypeptides with three-finger loop b-sheet structures. Previous reports have revealed that GAGs are potential targets of cardiotoxins and showed the specificity of binding of the toxins to various GAGs. (Vyas, et al., 1998) Previously, Our laboratory members confirmed the different binding behavior between cardiotoxins and heparin. However, the interaction between HMW-heparin and cardiotoxins displayed the higher degree of complexity. Here we adopted enzymatic depolymerization method to harvest the heparin fragments, which implicated differential templates of lengths for GAG-binding, for investigation of the binding phenomena. This thesis deals with the mode of association between cardiotoxins and GAGs, by intrinsic and extrinsic fluorescence spectroscopy. Furthermore, a comparison has been made for the variation in binding affinity of cardiotoxins towards various heparin fragments and different GAGs. The results showed that the cardiotoxin binding affinities of GAGs indicate the different binding modes of the interaction. The interaction between HMW-heparin and cardiotoxins reveals higher affinity behavior and may create hydrophobic domains for 1,8-ANS binding. The fluorescence spectroscopy was used to investigate the interaction between cardiotoxins and heparin fragment. The intrinsic fluorescence intensity revealed the different interactions between cardiotoxins and heparins, and the apparent change of intrinsic fluorescence intensity implicates distinctive modes of interaction. The extrinsic fluorescence intensity indicated that the accumulation of proteins on heparin promotes additional hydrophobic domains for ANS binding only when cardiotoxins interact with longer chains of heparin.In addition to this extrinsic fluorescence intensity of the interaction mode of different cardiotoxins, CTX Tg and CTX M1, with GAGs has also been compared. The difference between CTX Tg and CTX M1 may be due to the change in Asp to Asn. Therefore, possible ANS binding domain is near to Asn57 of CTX M1. The results showed that the hydrophobic domain will be formed from the interaction between cardiotoxins and GAGs, and the resultant complex would be different due to various reactants. It is concluded that the distinction of affinity modes from the interaction between cardiotoxins and GAGs are specific, and also the binding sites are different in these modes.