缢蛏碱性磷酸酶胍溶液中分子折叠与活力变化研究

Study on Relationship between the Molecular Folding and Activity of ALkaline Phosphatase from Sinonovacula Constricty in the Presence of Guanidine Hydrochloride

报道了缢蛏碱性磷酸酶（简称ＡＬＰ）经不同浓度盐酸胍处理时酶的分子构象所发生的变化以及酶变化和失活的动力学过程。在胍中酶荧光发射峰强度下降，紫外差光谱在２４６ｎｍ和２８５ｎｍ处出现２个负峰，ＣＤ谱中酶的α螺旋度下降，且随浓度增大，变化程度也加大。动力学研究表明，酶在０．５ｍｏｌ／Ｌ、１．０ｍｏｌ／Ｌ、２．０ｍｏｌ／Ｌ３．０ｍｏｌ／Ｌ、４．０ｍｏｌ／Ｌ盐酸胍中的变性速度常数分别为３．２１×１０^（－４）ｓ^（－１）、６．３８×１０^（－４）ｓ^（－１）、２．１７×１０^（－３）ｓ^（－１）、２．３３×１０^（－３）ｓ~９－１）、５．１７×１０^（－３）ｓ^（－１）；而酶在相应盐酸胍中的失活速度常数分别为２．３３×１０^（－４）ｓ^（－１）、３．５７×１０^（－４）ｓ^（－１）、５．８６×１０^（－４）ｓ^（－１）、１．１４×１０^（－３）ｓ^（－１）、３．４５×１０^（－３）ｓ^（－１）；表现为失活与构象伸展变化基本平行。

Effect of guanidine hydrochloride (GuHcl) on the changes in molecular conformation and activity, kinetics of denaturation and inactivation of alkaline phosphatase (ALP)from Sinonovacula constrica are reported in this essay. In the presence of guanidine, the flu rescence emission peak of ALP decreased; On the other hand, two negative peaks merged at the wavelengths of 246 nm and 285 nm on the UV differential spectra;and the αhelix degree in the circular dichroism spectra reduced. Denaturation rate constants of ALP in different concentrations of GuHCl (0. 5 , 1.0 , 2. 0 , 3. 0, 4. 0 mol/L) measured by fluorescence were 3. 21×10￣(-4), 6.38×10￣(-4) , 2. 17×10￣(-3) , 2.33×10￣(-3) and 5.16×10￣(-3)s￣(-1) ,respectively. Meanwhile the respective inactivation rate constants were 2. 33×10￣(-4) , 3.57 × 10￣(-4), 5. 86×10￣(-4), 1. 14×10￣(-3) and 3. 45×10￣(-3)s￣(-1). Results indicate that the rates of conformational changes of ALP in the presence of GuHCl are slightly faster than those of inactivation, though they all fall in the same order of magnitude. We propose to call such relationship the ″synchronous alteration type″ for rate changes in conformation and inactivation of the same order during denaturation.