High-pressure FTIR study of the stability of horseradish peroxidase. Effect of heme substitution, ligand binding, Ca++ removal, and reduction of the disulfide bonds.
High-pressure FTIR study of the stability of horseradish peroxidase. Effect of heme substitution, ligand binding, Ca++ removal, and reduction of the disulfide bonds.
复制标题
辣根过氧化物酶稳定性的高压 FTIR 研究。
作者：
L. Smeller;F. Meersman;J. Fidy;K. Heremans
介绍了辣根过氧化物酶同工酶 C 的压力稳定性以及可能的稳定因子的鉴定。通过 FTIR 光谱研究了血红素取代、Ca(2+) 去除、小底物分子（苯并异羟肟酸）的结合以及二硫键还原对压力稳定性的影响。发现 HRP 在高压下极其稳定，展开中点为 12.0 +/- 0.1 kbar。虽然用血红素替代不含金属的中卟啉并没有改变解折叠压力，但 Ca(2+) 去除和底物结合分别使解折叠中点降低了 2.0 和 1.2 kbar。脱辅基蛋白显示出高达 10.4 kbar 的转变。然而，在大气压下存在的折叠结构的量远低于所有其他形式的 HRP 中的折叠结构量。二硫键的还原导致压力稳定性最低的形式，展开中点为 9.5 kbar。然而，这仍然远高于蛋白质的平均压力稳定性。高压稳定性和压力引起的光谱变化分析表明，该蛋白质具有刚性核心，这导致了高稳定性，同时也存在稳定性较差和构象迁移性较高的区域。
The pressure stability of horseradish peroxidase isoenzyme C and the identification of possible stabilizing factors are presented. The effect of heme substitution, removal of Ca(2+), binding of a small substrate molecule (benzohydroxamic acid), and reduction of the disulfide bonds on the pressure stability were investigated by FTIR spectroscopy. HRP was found to be extremely stable under high pressure with an unfolding midpoint of 12.0 +/- 0.1 kbar. While substitution of the heme for metal-free mesoporphyrin did not change the unfolding pressure, Ca(2+) removal and substrate binding reduced the midpoint of the unfolding by 2.0 and 1.2 kbar, respectively. The apoprotein showed a transition as high as 10.4 kbar. However, the amount of folded structure present at the atmospheric pressure was considerably lower than that in all the other forms of HRP. Reduction of the disulfide bonds led to the least pressure stable form, with an unfolding midpoint at 9.5 kbar. This, however, is still well above the average pressure stability of proteins. The high-pressure stability and the analysis of the pressure-induced spectral changes indicate that the protein has a rigid core, which is responsible for the high stability, while there are regions with less stability and more conformational mobility.