Naderi Beni Ramin, Hassani-Nejad Pirkouhi Zahra, Mehraban Fouad, Seyedarabi Arefeh
Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran 1417614411, Iran.
ACS Omega. 2023 May 26;8(23):20714-20729. doi: 10.1021/acsomega.3c01288. eCollection 2023 Jun 13.
Major ozone autohemotherapy (MAH) is a popular clinical practice for treating a variety of pathological conditions due to the mild and controlled oxidative stress produced by the reaction of ozone gas with other biological components. Previous studies have shown that blood ozonation leads to structural changes in hemoglobin (Hb); therefore, in the present study, the molecular effects of ozonation on Hb of a healthy individual were assessed by ozonating whole blood samples with single doses of ozone at 40, 60, and 80 μg/mL or double doses of ozone at 20 + 20, 30 + 30, and 40 + 40 μg/mL ozone to investigate whether ozonating once versus twice (but with the same final ozone concentration) would have varying effects on Hb. Additionally, our study aimed to verify whether using a very high ozone concentration (80 + 80 μg/mL), despite mixing it with blood in two steps, would result in Hb autoxidation. The pH, oxygen partial pressure, and saturation percentage of the whole blood samples were measured through a venous blood gas test, and the purified Hb samples were analyzed using several techniques including intrinsic fluorescence, circular dichroism and UV-vis absorption spectroscopies, SDS-polyacrylamide gel electrophoresis, dynamic light scattering, and a zeta potential analyzer. Structural and sequence analyses were also used to study the Hb heme pocket autoxidation sites and the residues involved. The results showed that the oligomerization and instability of Hb can be reduced if the ozone concentration to be used in MAH is divided into two doses. Indeed, our study demonstrated that two-step ozonation with 20, 30, and 40 μg/mL of ozone instead of single-dose ozonation with 40, 60, and 80 μg/mL of ozone reduced the potential adverse effects of ozone on Hb including protein instability and oligomerization. Moreover, it was found that for certain residues, their orientation or displacement leads to the entry of excess water molecules into the heme moiety, which can contribute to Hb autoxidation. Additionally, the autoxidation rate was found to be higher in alpha globins compared to beta globins.
大自血臭氧疗法（MAH）是一种常用的临床治疗方法，用于治疗多种病理状况，这是因为臭氧气体与其他生物成分反应产生的氧化应激较为温和且可控。先前的研究表明，血液臭氧化会导致血红蛋白（Hb）的结构发生变化；因此，在本研究中，通过用40、60和80μg/mL的单剂量臭氧或20 + 20、30 + 30和40 + 40μg/mL的双剂量臭氧对全血样本进行臭氧化，评估了臭氧对健康个体Hb的分子效应，以研究单次臭氧化与两次臭氧化（但最终臭氧浓度相同）对Hb的影响是否不同。此外，我们的研究旨在验证，尽管将非常高的臭氧浓度（80 + 80μg/mL）分两步与血液混合，是否会导致Hb自氧化。通过静脉血气检测测量全血样本的pH值、氧分压和饱和度百分比，并使用包括内源荧光、圆二色性和紫外可见吸收光谱、SDS-聚丙烯酰胺凝胶电泳、动态光散射和zeta电位分析仪在内的多种技术对纯化的Hb样本进行分析。还使用结构和序列分析来研究Hb血红素口袋自氧化位点和相关残基。结果表明，如果将MAH中使用的臭氧浓度分为两剂，可以降低Hb的寡聚化和不稳定性。事实上，我们的研究表明，用20、30和40μg/mL的臭氧进行两步臭氧化，而不是用40、60和80μg/mL的臭氧进行单剂量臭氧化，可降低臭氧对Hb的潜在不利影响，包括蛋白质不稳定性和寡聚化。此外，发现对于某些残基，它们的取向或位移会导致过量水分子进入血红素部分，这可能导致Hb自氧化。此外，发现α珠蛋白中的自氧化速率高于β珠蛋白。
