Abstract
Aliphatic amines ( ε -amino group of Lys) are moderately nucleophilic when deprotonated (beyond pH 9 for the free amino acid, but often much lower in proteins); on the other hand, the inactivation of the labelling reagent by water increases with pH. The α -amino group has a pKa of 7. 6–8. 5, thus the N-terminus of a protein can be labelled specifically around pH 7. Aromatic amines require very reactive probes (thiocyanate, sulphonyl chloride), but can be labelled at any pH > 4. The nitrogen of peptide bonds and Gln, Asn, Arg and His side-chains, and of guanosine and adenosine are almost unreactive. Amino-sugars in glycoproteins may also react.
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Commercial preparations of sodium cyanoborohydride are often contaminated with interfering material. It should be re-crystallised before use (11g dissolved in 25ml acetonitrile, centrifuged and precipitated with 150ml methylene chloride over night at 4∘C). Store in vacuo and make fresh solutions every day. Nickel or cobalt ions may be used to trap cyanide during reduction with cyanoborohydride.
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© 2011 Springer Science+Business Media, LLC
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Buxbaum, E. (2011). Amine-Reactive Reagents. In: Biophysical Chemistry of Proteins. Springer, Boston, MA. https://doi.org/10.1007/978-1-4419-7251-4_19
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DOI: https://doi.org/10.1007/978-1-4419-7251-4_19
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