Abstract
Disulfide bonds in soybean trypsin inhibitor (Kunitz) were simultaneously reduced and alkylated using tri-n-butylphosphine and 2-vinylquinoline at pH 7.6 in 0.11 M Tris-4.4 M urea, 41% ethanol. The resulting S-β-2-quinolylethylated protein (2-QE-STI) has a new absorption peak at 315–318 nm. Its quinoline fluorescence can be excited above 310 nm independently of intrinsic protein fluorescence. Free 2-quinolylethylcysteine (2-QEC) shows unexpectedly weak fluorescence. Quinoline absorption in 2-QEC and 2-QE-STI changes with pH. The apparentpK values determined spectrophotometrically are near 5 for 2-QEC and 3 for 2-QE-STI. Fluorescence decreased with increasing pH and in the presence of chloride ions. Both structural and charge effects thus appear to influence the absorption and fluorescence of the quinoline group. Corrected fluorescence emission (excited at 316 nm) of neutral 2-QE-STI diluted in 0.1 N H2SO4 was directly proportional to concentration in the range 0.4–8 μm 2-QEC. The 2-QEC content of the protein derivative determined by UV absorption at pH 1.5 was in agreement with the expected value of four residues per mole. Fluorescence measurements ofS-2-quinolylethylated proteins may be especially useful as a sensitive, specific assay for cyst(e)ine residues.
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Reference to a company or product name does not imply approval or recommendation of the product by the U.S. Department of Agriculture to the exclusion of others that may be suitable.
Abbreviations used are Mops: 3-(N-morpholino)propanesulfonic acid; STI: soybean trypsin inhibitor (Kunitz); 2-PE-STI:S-β-2-pyridylethylated STI; 2-QEC:S-β-(2-quinolylethyl)-l-cysteine; 2-QE-STI:S-β-2-quinolylethylated STI; TosPheCH2-trypsin: bovine trypsin treated withp-toluenesulfonyl phenylalanine chloromethyl ketone.
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Zahnley, J.C., Friedman, M. Absorption and fluorescence spectra ofS-quinolylethylated Kunitz soybean trypsin inhibitor. J Protein Chem 1, 225–240 (1982). https://doi.org/10.1007/BF01025001
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DOI: https://doi.org/10.1007/BF01025001