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여러 가지 Perturbing Media에서의 알부민의 비가역적 열변성
윤성철,장광숙,Yoon, Sung-Chul,Chang, Kwang-Sook 생화학분자생물학회 1994 한국생화학회지 Vol.27 No.5
Two-state model을 바탕으로 하여 혈장 알부민(bovine serum albumin)과 그 disulfide 결합이 끊어진 cysteinyl-알부민의 비가역적 열변성 기작을 조사하였다. 고감도 시차주사 열량계(differential scanning calorimeter, DSC)로부터 얻은 알부민의 DSC scan profiles을 속도론적으로 분석하므로써 열변성 활성화에너지($E{\alpha}$)를 구하였다. Urea, N-methylurea, N,N'-dimethylurea, tetramethylurea, acetamide, thiourea, guanidine-HCl, sorbitol, glycerol 등과 같은 perturbants가 알부민의 변성에 어떤 영향을 주는지를 조사하였다. Perturbant의 종류 및 농도에 따라 $E{\alpha}$가 216~380 kJ/mol의 범위값을 나타냈다. 비환원 알부민의 $E{\alpha}$가 cysteinyl-알부민보다 높게 나타났다. 이는 17개의 disulfide 결함이 알부민의 안정화에 기여함을 의미한다. 이용된 perturbants는 네가지 type으로 분류되었다. 첫째, sorbitol, glycerol, 등은 농도를 증가시킴에 따라 알부민의 $E{\alpha}$와 $T_m$(endothermic peak temperature) 모두를 증가시킴으로써 강력한 안정화제임을 나타냈다. 둘째, urea, 메틸치환 urea, thiourea 등은 농도가 증가함에 따라 알부민 변성 활성화에너지($E{\alpha}$)와 $T_m$의 감소를 보임으로써 강력한 변성제임을 나타냈다. 셋째, acetamide는 농도가 증가함에 따라 $E{\alpha}$의 증가, $T_m$의 감소의 양면성을 보여주었다. 특히, acetamide의 경우 농도가 7~8M일 때 알부민의 열변성은 정확한 two-state 전이과정을 거치는 것으로 확인되었다. 넷째, guanidine-HCl은 ~1M 이하 농도에서는 안정화 효과를 보이다가 그 이상 농도에서는 변성제로 작용하는 현상을 보였다. The thermal denaturation mechanisms of bovine serum albumin (BSA) and its reduced form of cysteinyl-BSA in PBS buffer (pH 7.0, ionic strength = 0.1 or 0.2) were investigated on the basis of kinetic analysis in which irreversible two-state transition ($N{\rightarrow}D$) was assumed. Several perturbants such as urea, N-methylurea, N,N'-dimethylurea, tetramethylurea, acetamide, thiourea, guanidine-HCl, sorbitol and glycerol were employed to perturb the system and to see their effect on the thermal denaturation of BSA and disulfide-broken cysteinyl-BSA. The thermal transition parameters, $T_m$ (denaturation peak temperature), ${\Delta}H_{cal}$ (endothermic heat) and $C_p$ (heat capacity) of the two proteins were measured on a Setaram micro-DSC (differential scanning calorimeter) in the presence or absence of those perturbants. The activation energies ($E{\alpha}$) of thermal denaturation of BSA and cysteinyl-BSA were estimated from the analysis of DSC-scan profiles. The estimated activation energies ($E{\alpha}$) ranged from 216 to ~380 KJ/mol depending on the type and the concentration of the perturb ant employed. The activation energy of thermal denaturation of unreduced BSA is higher than that of cysteinyl-BSA, which means that the 17 disulfide bonds contribute to the stabilization of BSA. It was also found that urea, methyl substituted ureas and high-concentration guanidine-HCl destabilized BSA, but the polyhydroxyl perturbants of sorbitol and glycerol stabilized the protein, which was determined from both $T_m$ and $E{\alpha}$ data. However, increasing acetamide concentration caused a gradual decrease of $T_m$ and a gradual increase of $E{\alpha}$ of BSA. Thus, this indicates that the two determinants ($T_m$ and $E{\alpha}$) for thermal stability of proteins are independent of each other. Apparent deviation from the two-state mechanism of BSA thermal denaturation in acetamide-containing PBS buffer was estimated by calculating the van't Hoff enthalpies (${\Delta}H_{VH}$) and comparing them with the calorimetric enthalpies (${\Delta}H_{cal}$). An increase of acetamide concentration generally decreased the deviation from the two-state mechanism.
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여러 가지 Perturbing media 에서의 알부민의 비가역적 열변성
윤성철,장광숙 ( Sung Chul Yoon,Kwang Sook Chang ) 생화학분자생물학회 1994 BMB Reports Vol.27 No.5
The thermal denaturation mechanisms of bovine serum albumin (BSA) and its reduced form of cysteinylBSA in PBS buffer (pH 7.0, ionic strength=0.1 or 0.2) were investigated on the basis of kinetic analysis in which in-eversible two-state transition (N→D) was assumed. Several perturbants such as urea, N-methylurea, N,N`-dimethylurea, tetrnmethylurea, acetamide, thiourea, guanidine-HCl, sorbitol and glycerol were employed to perturb the system and to see their effect on the thermal denaturation of BSA and disulfide-broken cysteinyl-BSA. The thermal transition parnmeters, T_m (denaturation peak temperature), ΔH_(cal) (endothermic heat) and C_p (heat capacity) of the two proteins were measured on a Setaram micro-DSC (differential scanning calorimeter) in the presence or absence of those perturbants. The activation energies (E_a) of thermal denaturation of BSA and cysteinyl-BSA were estimated from the analysis of DSC-scan profiles. The estimated activation energies (E_a) ranged from 216 to ∼380 KJ/mol depending on the type and the concentration of the perturbant employed. The activation energy of thermal denaturation of unreduced BSA is higher than that of cysteinyl-BSA, which means that the 17 disulfide bonds contribute to the stabilization of BSA. It was also found that urea, methyl substituted ureas and high-concentration guanidine-HCl destabilized BSA, but the polyhydroxyl perturbants of sorbitol and glycerol stabilized the protein, which was determined from both T_m and E_a data. However, increasing acetamide concentration caused a gradual decrease of T_m and a grndual increase of E_a of BSA Thus, this indicates that the two determinants (T_m and E_a) for thermal stability of proteins are independent of each other. Apparent deviation from the two-state mechanism of BSA thermal denaturation in acetamide-containing PBS buffer was estimated by calculating the van`t Hoff enthalpies (ΔH_(VH)) and comparing them with the calorimetric enthalpies (ΔH_(cal)). An increase of acetamide concentration generally decreased the deviation from the two-state mechanism.
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