血紅蛋白 central-cavity 靜電結構效應之蛋白工程研究

過去對血紅蛋白異位調節效應的研究指出，在血紅蛋白四合體中央空腔的區域的靜電排斥效應對分子機能的調節如氧合時氯離子的鍵結和波爾效應，扮演著一個很重要的角色。 本研究焦點放在血紅蛋白中央空腔中，帶有正電殘基的其中一個位置， b82 Lysyl，來研究其正電離子效應與人類血紅蛋白氧合反應熱及四合體安定性的相關。針對這個目標，我們利用電腦做胺基酸序列排列比對和分子結構的模擬，選擇以 Asparaginyl 殘基 (Asn; N) 取代 Lysyl 殘基 (Lys,K) 。經基因重組合成一個單點突變的重組血紅蛋白 (rHbK82Nb)，再經過一連串管柱層析純化之重組血紅蛋白。其純度以可見光譜和血色素電泳鑑定，並以氧合平衡曲線的測定，膠質層析和氧化速率的分析檢視重組血紅蛋白之性能。實驗結果顯示 β82Lysyl 殘基所帶的正電性確實影響血紅蛋白氧合時的放熱反應與其四合體的安定性。另一方面，這也說明 Lysyl 殘基所帶的正電性，確保大部份的陸生脊椎動物在一般大氣壓力下，溫和的溫度變化 ( 如 1 atm, 0 ~35 )℃ ℃ 能有效輸送氧氣。本研究的結果吻合先前中央空腔因陰離子如 Cl- , PO43- 改變其正電性而影響血紅蛋白輸氧機能的研究，並提示了中央空腔電荷分佈對血紅蛋白四合體穩定及氧合放熱效應的影響，要了解中央空腔電荷分佈的機轉，則需更進一步研究。

A protein engineering study on the contribution of charged residues in the central-cavity structure of hemoglobin

Previous studies by others and this laboratory on the allosteric effect of hemoglobins have suggested that the electrostatic replusive cavity at the centerof tetrameric moiety plays a substantial role in regulating O2 transport properties, including chloride binding, and Bohr effect . In attempt to obtaina further insight into the contribution of charged residues to other allosteric properties , in this study we designed and synthesized a site directed mutant , b82 Lys-Asn , for examing influencs the tetrameric stability and enthalpitic change in O2 binding of human adult hemoglobin (HbA). Through a series chromatographic purifucation, the purity of rHbK82Nb was verified with both UV-Vis spectroscopic and electrophoretic analyses.By O2 equilibrium, gel permissionchromatography, and autooxidation analyses, we have investigated that the positively charge of b82 Lysyl residue is indeed involved in the exhothermic O2 binding characteristics and somewhat stabilize the tetrameric heme moiety of HbA. These results imply that the Lysyl β82 conserved in mostly land living vertebrate animal hemoglobins, is made for uptake O2 effectively in the enviroment of normal atmosphere with mild temperature changes, i.e. l atm 0 陡 35 .Resul℃ts obtained from this study are consist to the previously finding of the positively charge in the Central-cavity for small anions such as Cl- and PO43- ions, and further evident that the influence of electrostatic structure of the central-cavity in the temperature effect and stability of hemoglobin tetramers .