[Frontiers In Bioscience, Landmark, 24, 1085-1096, Jan 1, 2019]

Interactions between human hemoglobin subunits and peroxiredoxin 2

Qiang Ma1, Liang An1, Huifang Tian2, Jia Liu1, Liqiang Zhang3, Xiaojing Li1, Chunhua Wei1, Caixia Xie1, Huirong Ding2, Wenbin Qin1, Yan Su1

1Laboratory of Hemoglobin, Baotou Medical College, 014060, Baotou, China, 2Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Central Laboratory, Peking University Cancer Hospital & Institute, 100142, Beijing, China, 3State Key Laboratory of Heavy Oil Processing and Department of Materials Science and Engineering, China University of Petroleum, 102249, Beijing, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. IDQD prediction method
3.2. Preparation of RBC suspension and hemolysate
3.3. Purification of different Hb components from starch-agarose mixed gel
3.4. Coimmunoprecipitation (co-IP)
3.5. SDS-PAGE
3.6. In-gel digestion
3.7. Identification of proteins by liquid chromatography tandem mass spectrometry
3.8. Western blotting
3.9. XAFS experiment
4. Results
4.1. Discrimination of the interactions between human hemoglobin subunits and Prx2 based on the IDQD model
4.2. Comparison of IDQD-predicted interactions between different Hb subunits with the experimental results
4.3. Interaction between HbA and Prx2
4.4. XAFS to clarify the interaction mechanism between HbA and Prx2
5. Discussion
6. Acknowledgments
7. References

1. ABSTRACT

Red blood cells (RBCs) are exposed to exogenous reactive oxygen species in the circulatory system. To this end, the interactions between the different hemoglobin (Hb) subunits and peroxiredoxin 2, which is a ubiquitous member of the antioxidant enzymes that also controls the cytokine-induced peroxide levels, were assessed. We predicted by the increment of diversity with quadratic discriminant analysis (IDQD) that peroxiredoxin2 (Prx2) could interact with the hemoglobin alpha, beta and gamma subunits but not with the delta subunit. Coimmunoprecipitation (co-IP), electrospray ionization quadrupole time of flight (ESI-Q-TOF) mass spectrometry, Western blotting and X-ray absorption fine structure (XAFS) spectroscopy were performed to verify these predictions. The results showed that Prx2 was a member of the beta-globin immunoprecipitating complex that existed in hemoglobin A, hemolysate-hemoglobin A, hemoglobin A-hemoglobin A2, hemolysate-hemoglobin A-hemoglobin A2 and hemoglobin A2 but not in hemolysate-hemoglobin A2. Adding Prx2 to hemoglobin A altered the second shell of iron embedded in hemoglobin A. Therefore, Prx2 interacts with hemoglobin A (Alpha2Beta2) and hemoglobin F (Alpha2Gamma2) but not with hemoglobin A2 (Alpha2Delta2).

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Abbreviations: Reactive oxygen species (ROS), hemoglobin (Hb), peroxiredoxin2 (Prx2), increment of diversity with quadratic discriminant analysis (IDQD), co-immunoprecipitation (co-IP), electrospray ionization quadrupole-time of flight (ESI-Q-TOF), X-ray absorption fine structure (XAFS), protein-protein interactions (PPIs), support vector machines (SVMs), random forests (RFs), red blood cells (RBCs), reactive oxygen species (ROSs), glutathione peroxidase (GSH-Px), peroxiredoxins (Prxs), X-ray absorption near-edge structure (XANES), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), trifluoroacetic acid (TFA).

Key Words: Peroxiredoxin 2, Hemoglobin, Interaction, Increment Of Diversity With Quadratic Discriminant Analysis, X-Ray Absorption Fine Structure

Send correspondence to: Yan Su, Laboratory of Hemoglobin, Baotou Medical College, 014060, Baotou, Inner Mongolia, China, Tel: 86-472-7167834, Fax: 86-472-7167834, E-mail: synmg@126.com