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Covalent cross‐linking of proteins without chemical reagents
Author(s) -
Simons Brigitte L.,
King Mary C.,
Cyr Terry,
Hefford Mary Alice,
Kaplan Harvey
Publication year - 2002
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.4390102
Subject(s) - covalent bond , dimer , chemistry , molecule , rnase p , peptide bond , monomer , reagent , amide , lysozyme , macromolecule , stereochemistry , organic chemistry , enzyme , biochemistry , polymer , rna , gene
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.