Covalent cross-linking of proteins without chemical reagents.
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 degrees 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.
Simons
Brigitte L
BL
Centre for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Canada K1A 0L2.
King
Mary C
MC
Cyr
Terry
T
Hefford
Mary Alice
MA
Kaplan
Harvey
H
D024342:Amino Acids, Acidic / Q000737:chemistry; D024361:Amino Acids, Basic / Q000737:chemistry; D000818:Animals; D002417:Cattle; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005612:Freeze Drying; D006358:Hot Temperature; D013058:Mass Spectrometry; D008722:Methods; D009113:Muramidase / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry
D024342:Amino Acids, Acidic; D024361:Amino Acids, Basic; D011506:Proteins; D012259:Ribonuclease, Pancreatic; D009113:Muramidase