利用FT-ICR MS 探討蛋白質與輔基間的鍵結強度

Investigation of Binding Energy between Myoglobin and Heme

魏世嘉(Shih-Chia Wei)

指導教授 ： 李遠哲

共同指導教授 ： 張煥正

國立臺灣大學/理學院/化學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.00439

摘要

本實驗藉由結合FT-ICR MS與ESI兩項技術，並利用CAD兩種不同的激發技巧 (On-resonance excitation與SORI-CAD)。當作偵測Non-covalent bond 與Covalent bond絕對與相對強弱的工作。在本篇論文中，我們討論了：在不同pH值條件下，可觀測到氣相中的Myoglobin隨著pH值的減少而漸漸由Holomyoglobin變性成Apomyoglobin。同時我們藉由SORI-CAD的實驗，成功的觀測到Myoglobin與Heme含有兩種型式的鍵結強度；且知[Fe(II)-Heme]與Myoglobin peptide (Mb+10)間的鍵結較強於[Fe(III)-Heme]+與Myoglobin (Mb+9)的鍵結。另外，我們利用On-resonance的激發模式成功的測量[Fe(III)-Heme]+ → [Fe(III)-Heme]+-nCH2COOH• +nCH2COOH• (n=1 or 2)和hMb11+ → [Fe(III)-Heme]+ + aMb10+解離所需的能量。我們可以看到[Fe(III)-Heme]+其中的兩根Carboxyl methyl group隨著能量增加依序斷裂，其中所需能量大小依次為2.3±0.24 eV及2.9±0.27 eV。這樣的結果與DFT計算結果相比是一致的。
應用相同的技術，將hMb11+解離成[Fe(III)-Heme]+及aMb10+所需的ECM為1.0±0.12 eV。此結果與BIRD (利用改變腔體溫度)所得的活化能是一致的。同時也與其他研究團隊利用四極式質譜儀的高壓低能碰撞實驗相符。另外也觀察到hMbm+有Pseudo-native conformation及Denaturing conformation兩種構型。

關鍵字

傅立葉轉換質譜儀 ； 血紅素 ； 肌紅蛋白 ； 碰撞誘導解離

並列摘要

In this report, the structure and the binding characteristics of ferri-heme as well as its complex with myoglobin have been studied by using collision-induced dissociation (CID) method in a Fourier-transform ion cyclotron resonance (FT-ICR) cell. Performing on-resonance RF excitation to the selective precursor, we monitored the dependencies of product yields of CID as a function of ion energies and determined their dissociation thresholds. By using this method, we determined unambiguously that the heme, as a major oxygen-binding site of haemprotein, is only loosely bound to myoglobin with dissociation threshold of 1.0 ± 0.12 eV. Furthermore, an early dissociation started at 0.1 ± 0.03 eV suggesting the hMb ion contains two different conformers in our measurement. In applying it to the investigation of decomposition of Fe(III)-heme+, we determined that the decomposition of two carboxymethyl groups consecutively from it require threshold energies of respectively 2.3 ± 0.24 eV and 2.9 ± 0.27 eV. Moreover, the relative bond energy of its side chains is also obtained as: carboxymethyl groups < methyl groups < ethyl groups. Theoretical prediction carried out using density functional theory (DFT) method in both B3LYP/6-31G and B3LYP/6-311++G** level basis sets agree reasonably well with our observation. This method is unique in providing a rapid, non-selective, simple, and reliable qualitative measurement of dissociation energetics of both covalent and non-covalent interactions.

並列關鍵字

FT-ICR MS ； Heme ； Myoglobin ； CAD

參考文獻

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