Ivanova Irina A, Ershova Maria O, Shumov Ivan D, Valueva Anastasia A, Ivanov Yuri D, Pleshakova Tatyana O
Institute of Biomedical Chemistry, 119121 Moscow, Russia.
Biomedicines. 2022 Oct 20;10(10):2645. doi: 10.3390/biomedicines10102645.
This paper presents an investigation of the temperature dependence of the oligomeric state of the horseradish peroxidase (HRP) enzyme on the temperature of its solution, and on the solution storage time, at the single-molecule level. Atomic force microscopy has been employed to determine how the temperature and the storage time of the HRP solution influence its aggregation upon direct adsorption of the enzyme from the solution onto bare mica substrates. In parallel, spectrophotometric measurements have been performed in order to estimate whether the HRP enzymatic activity changes over time upon the storage of the enzyme solution. The temperature dependence of the HRP oligomeric state has been studied within a broad (15-40 °C) temperature range. It has been demonstrated that the storage of the HRP solution for 14 days does not have any considerable effect on the oligomeric state of the enzyme, neither does it affect its activity. At longer storage times, AFM has allowed us to reveal a tendency of HRP to oligomerization during the storage of its buffered solution, while the enzymatic activity remains virtually unchanged even after a 1-month-long storage. By AFM, it has been revealed that after the incubation of a mica substrate in the HRP solution at various temperatures, the content of the mica-adsorbed oligomers increases insignificantly owing to a high-temperature stability of the enzyme.
本文在单分子水平上研究了辣根过氧化物酶（HRP）的寡聚状态对其溶液温度及溶液储存时间的依赖性。利用原子力显微镜来确定HRP溶液的温度和储存时间如何影响酶从溶液直接吸附到裸云母基底上时的聚集情况。同时，进行了分光光度测量，以评估酶溶液储存时HRP的酶活性是否随时间变化。在较宽的温度范围（15 - 40°C）内研究了HRP寡聚状态的温度依赖性。结果表明，HRP溶液储存14天对酶的寡聚状态没有任何显著影响，也不影响其活性。在更长的储存时间下，原子力显微镜使我们能够揭示在其缓冲溶液储存期间HRP的寡聚化趋势，而即使经过1个月的储存，酶活性实际上仍保持不变。通过原子力显微镜发现，在不同温度下将云母基底在HRP溶液中孵育后，由于酶的高温稳定性，云母吸附的寡聚物含量增加不明显。
