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Selective fluorescent labeling at the α‐amino group of bovine pancreatic trypsin inhibitor
Author(s) -
Amir Dan,
Varshavski Lidya,
Haas Elisha
Publication year - 1985
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.360240404
Subject(s) - chemistry , fluorescence , trypsin , peptide , amino acid , covalent bond , yield (engineering) , absorption (acoustics) , derivative (finance) , biochemistry , enzyme , organic chemistry , physics , materials science , quantum mechanics , acoustics , financial economics , economics , metallurgy
Experimental investigation of protein structure and dynamics by spectroscopic methods using external probes requires attachment of a probe to a well‐defined 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 well‐defined sites. We have used specific protection of ε‐amino groups using tert‐butylazidoformate at high pH for covalent attachment of the fluorescent probe 2‐naphthoxyacetic acid at the α‐amino group of bovine pancreatic trypsin inhibitor (BPTI). The product is a chromatoraphically homogenous protein derivative that contains the probe at a dye to protein ratio of 1:1, specifically located at the N‐terminus, and 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 labled 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 monoexponetial fluorescence decay. These and other charactristics make this probe a good energy donor in transfer‐efficiency measurements.