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Covalent cross-linking of proteins without chemical reagents

¹ Centre for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Canada K1A 0L2
² Department of Chemistry University of Ottawa, Ottawa, Ontario K1N 6N5

Reprint requests to: Harvey Kaplan, Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N 6N5; e-mail: hkaplan{at}uottawa.ca; fax: (613) 562-5180.

A facile method for the formation of zero-length covalent cross-links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross-linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85°C for 24 h. Under these conditions, approximately one-third of the total protein present becomes cross-linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero-length cross-links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross-linked dimer has only one amide cross-link and retains the enzymatic activity of the monomer. The in vacuo cross-linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross-linking, and co-lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross-linked dimer.

Keywords: Protein cross-linking; lyophilized protein; zero-length cross-linking; covalent cross-linking; in vacuo modification

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