冷冻保护剂对马和人血红蛋白分子热力学及动力学参数影响的比较分析

Comparative analysis of cryoprotective agents influence on thermodynamic and kinetic parameters of equine and human hemoglobin molecules.

作者信息

Zinchenko Aleksandra V, Govorova Yuliana S

机构信息

Department of Cryobiophysics, Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, Kharkov, Ukraine.

出版信息

Cryo Letters. 2014 Nov-Dec;35(6):516-20.

PMID:25583012

Abstract

BACKGROUND

Critical to the understanding the mechanism of destruction and protection during cryopreservation of biological objects is the knowledge of the conformational transitions of biopolymers experiencing low temperatures in the presence of cryoprotective agents. This information may be derived from the kinetic and thermodynamic parameters of macromolecular thermal denaturation kinetics under different environmental conditions.

OBJECTIVE

The study deals with the influence of cryoprotective agents (glycerol, 1.2-propanediol (1.2-PD), and dimethyl sulfoxide (DMSO)) on thermodynamic and kinetic parameters of equine and human hemoglobin.

METHODS

Thermograms were recorded with differential scanning adiabatic microcalorimeter (DASM-4, Biopribor, Russia).

RESULTS

Temperatures and enthalpy changes in the denaturation of hemoglobins (ΔHcal) in the presence of cryoprotective agents from 0-40% (w/w) were determined. Within the whole concentration range glycerol slightly alters the hemoglobin denaturation temperature while DMSO and 1.2-PD lead to a significant decrease. The addition of cryoprotectants to hemoglobin solutions induces a complex behavior of ΔHcal concentration dependences of denaturation caused by intra- and intermolecular processes such as hydration of the porphyrin cycle, heme cleavage from heme-containing proteins, disorder in hydrophobic contacts with globin etc. These factors may decrease thermal stability by loosening hemoglobin molecules and spatial disruption of fragments of the protein. Activation energy of irreversible unfolding of equine hemoglobin at heating was estimated using the approach of Sanchez-Ruiz et al. DMSO and 1.2-PD decreased activation energy values.

CONCLUSIONS

The data indicate the reduction of protein thermal stability by DMSO and 1.2-PD. Glycerol slightly increases hemoglobin thermal stability within the studied range of concentrations.

摘要

背景

了解生物物体冷冻保存过程中的破坏和保护机制，关键在于掌握生物聚合物在低温及冷冻保护剂存在下的构象转变。该信息可从不同环境条件下大分子热变性动力学的动力学和热力学参数中获取。

目的

研究冷冻保护剂（甘油、1,2 - 丙二醇（1,2 - PD）和二甲基亚砜（DMSO））对马和人血红蛋白的热力学和动力学参数的影响。

方法

使用差示扫描绝热微量热计（DASM - 4，俄罗斯Biopribor公司）记录热谱图。

结果

测定了在0 - 40%（w/w）冷冻保护剂存在下血红蛋白变性的温度和焓变（ΔHcal）。在整个浓度范围内，甘油对血红蛋白变性温度的影响较小，而DMSO和1,2 - PD则导致显著降低。向血红蛋白溶液中添加冷冻保护剂会引发ΔHcal浓度依赖性变性的复杂行为，这是由分子内和分子间过程引起的，如卟啉环的水合作用、含血红素蛋白质中血红素的裂解、与珠蛋白疏水接触的紊乱等。这些因素可能通过使血红蛋白分子松弛和蛋白质片段的空间破坏来降低热稳定性。使用Sanchez - Ruiz等人的方法估算了马血红蛋白加热时不可逆展开的活化能。DMSO和1,2 - PD降低了活化能值。