Development of a System for Biosynthesis, Isolation and Purification of Holoform of Recombinant Human Neuroglobin and Its Characteristics

M. A. Semenova; Семенова М. А.; D. A. Dolgikh; Долгих Д. А.; M. P. Kirpichnikov; Кирпичников М. П.; G. V. Maksimov; Максимов Г. В.; N. A. Brazhe; Браже Н. А.; E. V. Bocharov; Бочаров Э. В.; R. H. Ziganshin; Зиганшин Р. Х.; E. Y. Parshina; Паршина Е. Ю.; A. A. Ignatova; Игнатова А. А.; O. M. Smirnova; Смирнова О. М.; Z. V. Bochkova; Бочкова Ж. В.; R. V. Chertkova; Черткова Р. В.

doi:10.31857/S013234232303020X

Development of a System for Biosynthesis, Isolation and Purification of Holoform of Recombinant Human Neuroglobin and Its Characteristics

Authors: Semenova M.A.¹, Dolgikh D.A.¹^,2, Kirpichnikov M.P.¹^,2, Maksimov G.V.³, Brazhe N.A.³, Bocharov E.V.¹, Ziganshin R.H.¹, Parshina E.Y.³, Ignatova A.A.¹, Smirnova O.M.¹, Bochkova Z.V.¹^,3, Chertkova R.V.¹
Affiliations:
1. Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, RAS
2. Biological Faculty, Lomonosov Moscow State University
3. Biophysics Department, Biological Faculty, Lomonosov Moscow State University
Issue: Vol 49, No 3 (2023)
Pages: 319-330
Section: Articles
URL: https://archivog.com/0132-3423/article/view/670627
DOI: https://doi.org/10.31857/S013234232303020X
EDN: https://elibrary.ru/PEAUKB
ID: 670627

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Abstract
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Abstract

An efficient system for the biosynthesis, isolation and purification of recombinant human neuroglobin has been developed and optimized, which makes it possible to produce protein in quantities sufficient to study its properties. According to UV-visible, IR-, CD-, and NMR spectroscopy data, recombinant neuroglobin is a structured protein in the holoform state. The data of chromato-mass-spectrometric analysis made it possible to conclude that there is a correctly formed disulfide bond in the structure of the oxidized form of the protein. Using Raman and surface-enhanced Raman spectroscopy with laser excitation at 532 nm, it was shown that heme in the reduced and oxidized forms of neuroglobin has vibrational degrees of freedom typical of b-type hemes, and the iron atom is six-coordinated. Using Raman spectroscopy with laser excitation at 633 nm, it was found that reduced –SH-groups were present in reduced neuroglobin, while in oxidized neuroglobin disulfide bridge was formed. The results obtained serve as the basis for detailed studies of the mechanism of the functioning of neuroglobin as a neuroprotector, in particular, during its interaction with oxidized cytochrome c, which is released from mitochondria in violation of their functioning and/or morphology.

Keywords

neuroglobin, recombinant proteins, heme-containing proteins, globins

References

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