Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed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)

How to cite: Karnaukhova, E.; Owczarek, C.; Schmidt, P.; Schaer, D.J.; Buehler, P.W. Human Plasma and Recombinant Hemopexins: Heme Binding Revisited. Preprints 2020, 2020120370 (doi: 10.20944/preprints202012.0370.v1). Karnaukhova, E.; Owczarek, C.; Schmidt, P.; Schaer, D.J.; Buehler, P.W. Human Plasma and Recombinant Hemopexins: Heme Binding Revisited. Preprints 2020, 2020120370 (doi: 10.20944/preprints202012.0370.v1).

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.

Subject Areas

Heme; Hemopexin; Circular dichroism; Recombinant hemopexin

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