山葵過氧化酶的摺疊與未折疊性質

Folding and Unfolding Properties of Horseradish Peroxidase.

廖千瑋(Chien-Wei Liao)

指導教授 ： 張一知

國立臺灣師範大學/理學院/化學系/碩士(2008年)

摘要

摘 要
本實驗研究山葵過氧化酶 (Horseradish Peroxidase, HRP) 之摺疊與其和催化活性的關聯性。HRP 摺疊程度的變化可利用不同濃度胍鹽酸 (guanidine hydrochloride) 下的 α-helix 含量推算而得。在胍鹽酸 5 ~ 7 M 之間，α-helix 含量有明顯變化，胍鹽酸提供 HRP 傾向 unfolding 的驅動力，以致其濃度越增大時，使 HRP 結構越趨鬆散，而摺疊與變性兩狀態間的 ΔG° 越小，且胍鹽酸濃度與 ΔG° 呈線性關係，由外插法可求出 25.0 ℃ 無胍鹽酸存在下的 HRP 之 ΔG° 為 60.84 kJ/mol。HRP 催化活性可由測量在 H2O2 的存在下催化 phenol 的反應而得。由數據分析可得 HRP 的 Km 與 Vmax 值分別為 2.321 mM 與 0.116 ΔA/s。催化反應中，Km (mM) 與 Vmax (ΔA/s) 之值在胍鹽酸存在下 (括弧中為其濃度) 分別為 4.734、0.151 (0.25 M)，4.222、0.128 (0.5 M)，3.128、0.113 (1 M)，3.083、0.104 (3 M)，9.539、0.147 (5 M)，19.928、0.152 (5.5 M)，391.26、0.907 (6 M)。由實驗結果顯示，HRP 的活性在加入 0.25 M 和 0.5 M 的微量胍鹽酸，在催化上有略高於正常摺疊的 HRP 活性。
加入低濃度 (～ 0.25 M) 的胍鹽酸，即使不會使 HRP 的 α-helix 含量改變，但催化活性略有增加，推測在低濃度胍鹽酸下，雖不使 HRP 結構瓦解，但構型上有些微影響，造成受質小分子較不易進入活化中心 (Km 增大)，但也因此變化造成反應速率較快 (Vmax 增大)，使得全反應變快。

關鍵字

山葵過氧化酶 ； 摺疊

並列摘要

Abstract
The relationship between folding and enzyme activity of Horseradish Peroxidase (HRP) has been studied. The folding conditions were deduced by the α-helix content of HRP at various guanidine hydrochloride (GuHCl) concentration. Circular Dichroism data showed that the α-helix content of HRP is changing dramatically between 5 ~ 7 M of GuHCl. The existence of GuHCl provides HRP the driving force toward unfolding. As GuHCl concentration increases, the difference of Gibbs energy of folding and unfolding HRP, (ΔΔG°), increases. Reaction ΔG° showed a linear relationship with GuHCl concentration and can be extrapolated to zero GuHCl concentration of ΔG° equals 60.84 kJ/mol at 25.0 ℃. Enzyme activity of HRP is determined from the reaction of phenol and H2O2. Enzyme catalysis values, Km and Vmax, of HRP are determined as 2.321 mM and 0.116 ΔA/s, respectively. In the denatured condition, Km (mM) and Vmax (ΔA/s) are determined in various GuHCl concentration (shown in parentheses) as 4.734, 0.151 (0.25 M); 4.222, 0.128 (0.5 M); 3.128, 0.113 (1 M); 3.083, 0.104 (3 M); 9.539, 0.147 (5 M); 19.928, 0.152 (5.5 M); 391.26, 0.907 (6 M) respectively. These results show that the enzyme activity of HRP in
0.25 M and 0.5 M GuHCl is higher than that of native HRP.
In low concentration of GuHCl (～ 0.25 M), there is no evidence of α-helix content changes, but the enzyme activity has slightly increased. These results indicate that though the α-helix content has not changed much in low GuHCl concentration, the protein conformation may change slightly. This conformational change is unfavor for substrate binding, but enhances reaction
rate. The overall effect is improved catalytic behavior.

並列關鍵字

Horseradish Peroxidase ； folding

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