Article
Version 1
Preserved in Portico This version is not peer-reviewed
Human Plasma and Recombinant Hemopexins: Heme Binding Revisited
Version 1
: Received: 14 December 2020 / Approved: 15 December 2020 / Online: 15 December 2020 (10:24:49 CET)
A peer-reviewed article of this Preprint also exists.
Karnaukhova, E.; Owczarek, C.; Schmidt, P.; Schaer, D.J.; Buehler, P.W. Human Plasma and Recombinant Hemopexins: Heme Binding Revisited. Int. J. Mol. Sci. 2021, 22, 1199. Karnaukhova, E.; Owczarek, C.; Schmidt, P.; Schaer, D.J.; Buehler, P.W. Human Plasma and Recombinant Hemopexins: Heme Binding Revisited. Int. J. Mol. Sci. 2021, 22, 1199.
Abstract
Abstract: Plasma hemopexin (HPX) is the key antioxidant protein of the endogenous clearance pathway that limits the deleterious effects of heme released from hemoglobin and myoglobin. During intra-vascular hemolysis, heme partitioning to protein and lipid increases as the plasma concentration of HPX declines. Therefore, the development of HPX as a replacement therapy during high heme stress could be a relevant intervention for hemolytic disorders. A logical approach to enhance HPX yield involves recombinant production strategies from human cell lines. The present study focuses on a biophysical assessment of heme binding to recombinant human HPX (rhHPX) produced in the Expi293FTM (HEK293) cell system. In this report, we examine rhHPX in comparison with plasma HPX using a systematic analysis of protein structural and functional characteristics related to heme binding. Analysis of rhHPX by UV/Vis absorption spectroscopy, circular dichroism (CD), SEC-HPLC and catalase-like activity demonstrated a similarity to HPX fractionated from plasma. In particular, the titration of HPX apo-protein(s) with heme was performed for the first time using a wide range of heme concentrations to model HPX-heme interactions to approximate physiological conditions (from extremely low to more than 2-fold heme excess). The CD titration data showed an induced bisignate CD Soret band pattern typical for plasma and rhHPX versions at low heme-to-protein molar ratios and demonstrated that further titration is dependent on the amount of protein-bound heme to the extent that the arising opposite CD couplet results in a complete inversion of the observed CD pattern. The data generated in this study suggests more than one binding site in both plasma and rhHPX. Further, our study provides a useful analytical platform for detailed characterization of HPX-heme interactions and potentially, novel HPX fusion constructs.
Keywords
Heme; Hemopexin; Circular dichroism; Recombinant hemopexin
Subject
Chemistry and Materials Science, Analytical Chemistry
Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Comments (0)
We encourage comments and feedback from a broad range of readers. See criteria for comments and our Diversity statement.
Leave a public commentSend a private comment to the author(s)
* All users must log in before leaving a comment