Covalent cross‐linking of proteins without chemical reagents

Abstract

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.

Application of zero-length cross-linking to form lysozyme, horseradish peroxidase and lysozyme–peroxidase dimers: Activity and stability

“…The in vacuo cross-linking procedure was performed by the method of Simons et al [15]. Lyophilized proteins were obtained from the supplier, reconstituted in distilled water to a concentration of 10 mg/ml for lysozyme and 5 mg/ml for peroxidase and then dialyzed against 0.2 M K 2 HPO 4 containing 0.15 M KCl at pH 6.5 for lysozyme and 0.02 M K 2 HPO 4 at pH 7 for peroxidase.…”

“…It can therefore provide some information about local protein-protein interactions and also has the advantage that no foreign group is introduced into the protein. The in vacuo cross-linking provides a novel and convenient procedure for achieving such cross-linking as reported by Simons et al [15]. However, they did not report any information about the activity and stability of the products.…”

“…specifically cross-linked hemoglobins are useful for the investigation of structure-function relationships [9][10][11], as well as in the development of potential substitutes for red blood cell transfusion [12][13][14]. Zero-length cross-linking is defined when the peptide chains are covalently linked through existing functional groups without incorporation of a spacer group [15]. This type of cross-linking has rarely been achieved and is most likely to occur when carboxyl groups are situated in proximity to amino groups, perhaps in salt linkages [15,16].…”

“…Zero-length cross-linking is defined when the peptide chains are covalently linked through existing functional groups without incorporation of a spacer group [15]. This type of cross-linking has rarely been achieved and is most likely to occur when carboxyl groups are situated in proximity to amino groups, perhaps in salt linkages [15,16]. It can therefore provide some information about local protein-protein interactions and also has the advantage that no foreign group is introduced into the protein.…”

A review on protein oligomerization process

“…Covalent protein oligomerization can also be carried out without the additional cross-linking agent by sealing a lyophilized protein in a vacuum at high temperature (<85 o C) for 24~96 hours. 33 Dimers, trimers, and traces of tetramers of ribonuclease A (RNase A) and lysozyme can be made without introducing chemical groups that could negatively affect the protein residues. In fact, the heat-vacuum treatment of proteins induces the dehydration of some of the lysine and aspartic acid or glutamic acid side-chains, producing newly formed intermolecular isopeptide bonds.…”