Use of the pH memory effect in lyophilized proteins to achieve preferential methylation of alpha-amino groups.
It is demonstrated that the pH memory effect can be used to control the ionization state of amino groups in lyophilized proteins and hence their chemical reactivity toward modifying reagents. When proteins were lyophilized from aqueous solutions at pH values between 6 and 7 and reacted in vacuo with iodomethane, the alpha-amino groups were found to be either preferentially or selectively trimethylated. Reaction with 13C-labeled iodomethane permitted detection and identification of individual trimethylated alpha-amino groups by 13C-NMR spectroscopy as distinct peaks in the spectral region between 52 and 57 ppm. There was adequate sensitivity to detect minor resonances of free alpha-amino groups arising from proteolysis of the major protein or from protein impurities. The resonances of the trimethylated alpha-amino groups in standard amino acids and peptides are sufficiently close to those in the derivatized protein to make a tentative identification of the N-terminal amino acid. It is also demonstrated that advantage can be taken of the pH memory effect to use the preferential 13C-methylation of amino groups to verify whether a protein has a free or blocked amino terminus.
Vakos
H T
HT
Department of Chemistry, University of Ottawa, Ontario, Canada.
Kaplan
H
H
Black
B
B
Dawson
B
B
Hefford
M A
MA
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002250:Carbon Radioisotopes / Q000737:chemistry; D002417:Cattle; D004798:Enzymes / Q000737:chemistry*; D005612:Freeze Drying / Q000379:methods*; D006847:Hydrocarbons, Iodinated / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine / Q000737:chemistry; D008745:Methylation; D019906:Nuclear Magnetic Resonance, Biomolecular; D012972:Sodium Hydroxide / Q000737:chemistry; D013696:Temperature
D000596:Amino Acids; D002250:Carbon Radioisotopes; D004798:Enzymes; D006847:Hydrocarbons, Iodinated; D019811:Hydroxylamine; D012972:Sodium Hydroxide; C014055:methyl iodide