Selective fluorescent labeling at the α-amino group of bovine pancreatic trypsin inhibitor

✍ Scribed by Dan Amir; Lidya Varshavski; Elisha Haas

• Publisher: Wiley (John Wiley & Sons)
• Year: 1985
• Tongue: English
• Weight: 812 KB
• Volume: 24
• Category: Article
• ISSN: 0006-3525
• DOI: 10.1002/bip.360240404

✦ Synopsis

Experimental investigation of protein structure and dynamics by spectroscopic methods using external probes requires attachment of a probe to a welldefined site and preparation of pure samples. Measurements of efficiency of nonradiative excitation energy transfer can yield very detailed information about the structure of proteins, provided that two different probes are selectively attached to welldefined sites. We have used specific protection of €-amino groups using tert-butylazidoformate at high pH for covalent attachment of the fluorescent probe 2-naphthoxyacetic acid at the a-amino group of bovine pancreatic trypsin inhibitor (BPTI). The product is a chromatographically homogenous protein derivative that contains the probe at a dye to protein ratio of 1:1, specifically located at the N-terminus, and that retains its full biological activity. The HPLC tryptic peptide map of BPTI has been analyzed, and all the peptide fragments have been identified. Analysis of tryptic fragments of the labeled BPTI derivative showed that it was selectively labeled at the N-terminal amino acid. The probe absorbs in the 310-325-nm range, which is spectrally distinct from the absorption of the protein, and has a monoexponential fluorescence decay. These and other characteristics make this probe a good energy donor in transferefficiency measurements.