Recombinant human erythropoietin (rHuEPO): Cross‐linking with disuccinimidyl esters and identification of the interfacing domains in EPO

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Abstract
Several amino groups of recombinant human erythropoietin are selectively cross‐linked by specific cross‐linkers including disuccinimidyl suberate or dithiobis(succinimidyl propionate). Intramolecular cross‐linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the cross‐linkers, which possess an 11–12‐Å length of a spacer between two reacting groups. Intramolecularly cross‐linked peptides obtained suggest that several amino groups in erythropoietin (EPO) are positioned at a distance of near 12 Å in the solution state. These interfacing amino groups include Lys 20–Lys 154, Lys 45–Lys 140, Lys 52–Lys 154, Lys 52–Lys 140, and Ala 1–Lys 116. A comparison of the cross‐linking results between nonglycosylated EPO and glycosylated EPO suggests that both proteins retain high similarity regarding protein conformation. These results fit a structural model similar to that of human growth hormone, in which four α‐helical bundles and a long stretch of β‐sheet structure are involved in the active protein.