□□構形與肝素結合之特定性研究｜國立清華大學博碩士論文庫

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研究生：	吳丞偉 Wu Cheng-Wei
論文名稱：	□□構形與肝素結合之特定性研究 Would the conformation of heparin-binding peptide play a possible role in heparin binding
指導教授：	呂平江博士 Lyu Ping-Chiang
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2000
畢業學年度：	88
語文別：	英文
論文頁數：	82
中文關鍵詞：	肝素、八糖肝素、醣胺素、圓二色旋光儀、螢光光譜儀、低分子量肝素、硫酸乙醯肝素、硫酸皮膚素
外文關鍵詞：	heparin, heparin octasaccharide, glycosaminoglycan, circular dichroism spectroscopy, fluorescence spectroscopy, LMWH (low molecular weight heparin), HS (heparian sulfate), DS (dermatan sulfate)
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中文摘要
(I)

肝素結合□□之構形的不同，是否會影響它與肝素的結合性質? 為了探討此點，便合成了稱之為 ApaK (O) 的□□，它的一級序列為 NH2-CNCKAPETAKCAKQCQKAQKAQAKQAKQW-NH2，由於ApaK (O)得以形成兩個雙硫鍵，因而便可形成α-helical 之構形，而另一□□，由於不能形成雙硫鍵，因此即為無特定構形，稱之為ApaK6 (R)，它們皆可與肝素結合，且被肝素誘導而增加α-helical 構形的程度。但一般的肝素皆為高度混合物，因此進一步的純化，是有必要的。且為了配合這29個胺基酸，因而純化得到了八糖肝素，且再進一步的純化，而得到八糖之中最高負電荷且存在最大量之物，稱之為octasaccharide Ⅷ。

當使用此octasaccharide Ⅷ 與 ApaK6 (O) 和 ApaK6 (R) 作用，觀察到一些現象:

1. 在生理條件之下，ApaK6 (O) 和 ApaK6 (R) 與octasaccharide Ⅷ 的解離常數，一者為202.5 μM，另一者為 497.8 μM。

2. 在結合時的結合量方面，octasaccharide Ⅷ 可提供 ApaK6 (O) 兩個結合量，但卻只提供ApaK6 (R) 一個結合量。

3. 由鹽濃度與解離常數之關係，求出ApaK6 (O) 與octasaccharide Ⅷ 作用時，約有四個靜電作用，而ApaK6 (R) 則約有五個靜電力。此外，在非靜電作用方面，前者的 DGnonionic 為 -10.2 KJ/mol，而後者為 -4.8 KJ/mol。所謂的非靜電作用，指的即為氫鍵力、凡得瓦力、和疏水性作用方面。由以上的結果，可以說在這個系統中，□□的構形對於肝素的結合，會扮演一定的角色。這或許可以用來解釋，在生物體內的肝素結合蛋白，於其上的肝素結合區域，有著不同的構形，而這不同的構形即代表了一定的意義。

4. 另外，除了使用octasaccharide Ⅷ 外，其它不同負電荷程度的八糖肝素，亦用來與這兩個□□測解離常數。而觀測到一有趣的現象，即最高負電荷的八糖肝素，並不見得是結合力最強的，這或許可以說負電荷的程度，並不是唯一決定結合力的因素。

(II)

為了測試與各種glycosamionglycans作用的程度，而設計出稱之為K828 的□□，它的一級序列為 SKAQKAQAKQAKQAQKAQKAQAKQAKQW-CONH2，在經由圓二色旋光儀和螢光光譜的結果顯示，此□□會趨向於對含有高度iduronic acid 的 glycosaminoglcans作用，如 LMWH、HS、和 DS，且作用後會增加α-helical 構形的程度，而結合的強度與α-helical 構形增加的程度是呈正相關的。

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.

Tables of Contents
ABSTRACT

INTRODUCTION

Part I

……………………………………………………………………………1

Figure 1.1 The repeating unit of heparin disaccharide

……………………………………………………………………………3

Figure 1.2 Three-dimensional structure of dodecasaccharide heparin

……………………………………………………………………………4

Figure 1.3 The helical wheel of K621

……………………………………………………………………………7

Figure 1.4 The helical wheel of A124-D145 sequence of D helix of antithrombin

……………………………………………………………………………11

Part II

……………………………………………………………………………13

Figure 2.1 The chemical structures of glycosaminoglycan family

……………………………………………………………………………14

Table 2.1 Amino Acid Composition of K828

……………………………………………………………………………17

Figure 2.2 The helica wheel of K828

……………………………………………………………………………18

Experimental Procedures

……………………………………………………………………………19

Result and Discussion

Part I

……………………………………………………………………………26

Figure 1.5 The circular dichroism spectrum of ApaK6 (O)

……………………………………………………………………………27

Figure 1.6 The circular dichroism spectrum of the ApaK6 (O)-LMWH complex

……………………………………………………………………………28

Figure 1.7 The circular dichroism spectrum of ApaK6 (R)

……………………………………………………………………………29

Figure 1.8 The circular dichroism spectrum of the ApaK6 (R)-LMWH complex

……………………………………………………………………………30

Figure 1.9 The plot of gel permeation chromatography

……………………………………………………………………………32

Figure 1.10 Changes in helix percentage of ApaK6 (O) as a function of added heparin oligosaccharide

……………………………………………………………………………34

Figure 1.11 Changes in helix percentage of ApaK6 (R) as a function of added heparin oligosaccharide

……………………………………………………………………………35

Figure 1.12 Anion exchanger elution profiles for heparin octasaccharide

……………………………………………………………………………37

Figure 1.13 The dissociation constant of Apak6 (O) binding with different sulfation patterns of heparin octasaccharide

……………………………………………………………………………40

Table 1.2 The dissociation constant of ApaK6 (O) complexed with the different sulfation components of heparin octasaccharide

……………………………………………………………………………41

Table 1.3 The dissociation constant of ApaK6 (R) complexed with the different sulfation components of heparin octasaccharide

……………………………………………………………………………42

Figure 1.14 The dissociation constant of Apak6 (R) binding with different sulfation patterns of heparin octasaccharide

……………………………………………………………………………43

Figure 1.15 Changes in the mean residue ellipticity of ApaK6 (R) at 222 nm upon octasaccharide Ⅷ titration

……………………………………………………………………………44

Figure 1.16 Changes in the mean residue ellipticity of ApaK6 (O) at 222 nm upon octasaccharide Ⅷ titration

……………………………………………………………………………45

Table 1.4 Comparied of the interaction of ApaK6 (O) and ApaK (R) with octasaccharide Ⅷ

……………………………………………………………………………46

Figure 1.17 Influence of ionic strength on the dissociation constant of the ApaK6 (O)-heparin octasaccharide Ⅷ  complex

……………………………………………………………………………52

Figure 1.17 Influence of ionic strength on the dissociation constant of the ApaK6 (R)-heparin octasaccharide Ⅷ  complex

……………………………………………………………………………53

Figure 1.19 vant Hoff plot of the ApaK6 (O)- heparin octasaccharide Ⅷ  complex

……………………………………………………………………………55

Figure 1.19 vant Hoff plot of the ApaK6 (R)- heparin octasaccharide Ⅷ  complex

……………………………………………………………………………56

Table 1.5 The statistics of the energy terms

……………………………………………………………………………58

Table 1.6 The statistics of the energy terms

……………………………………………………………………………58

Figure 1.20 One docking model of two ApaK6 (O) to one full sulfation heparin octasaccharide

……………………………………………………………………………59

Figure 1.21 The other docking model of two ApaK6 (O) to one full sulfation heparin octasaccharide

……………………………………………………………………………60

Conclusion

Part I

……………………………………………………………………………61

Result and Discussion

Part II

……………………………………………………………………………63

Figure 2.3 Plot of absorption values at 350 nm against concentration of LMWH

……………………………………………………………………………65

Figure 2.4 The circular dichroism spectrum of K828 in the absence and presence of various concentrations of LMWH

……………………………………………………………………………67

Figure 2.5 The changes in the mean residue ellipticity values of K828 upon titrating with CSA, DS, CSC, HA, and LMWH

……………………………………………………………………………70

Figure 2.6 Ring conformation of heparin monosaccharide

……………………………………………………………………………71

Figure 2.7 Changes in fluorescence anisotropy values of K828 upon titrating with CSA, DS, CSC, HA, and LMWH

……………………………………………………………………………74

Figure 2.8 Changes in the unfolded fractions of satureated K828-glycosaminoglycan complexes upon thermal denaturation

……………………………………………………………………………76

Concluson

Part II

……………………………………………………………………………77

Reference

……………………………………………………………………………78

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