Fluorescent tracer method for protein SH groups: III. Use ofN-(7-dimethylamino-4-methyleoumarinyl) malelmide as a tracer of cysteine-containing peptides
✍ Scribed by Keiichi Yamamoto; Takamitsu Sekine
- Book ID
- 102985209
- Publisher
- Elsevier Science
- Year
- 1978
- Tongue
- English
- Weight
- 731 KB
- Volume
- 90
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
We studied whether N-(7-dimethylamino-Cmethylcoumarinyl) maleimide (DACM) could be used as a fluorescent tracer for the purification and analysis of cysteinecontaining peptides. An addition product of DACM with SH compound was stable at room temperature in the solution of pH 9.0 and in 50% acetic acid. However, it was hydrolyzed when heated at 110°C for 48 h in 6 N HCl. On filter paper, 1 pmol of the addition product in a spot 3 mm in diameter was visible under ultraviolet illumination. The addition product was also stable on filter paper for at least several months after spotting. The elution velocity of the addition products with low molecular weight SH compounds in the Sephadex G-25 column was low considering their molecular weights. However, in general, the elution velocity increased with an increase in the molecular weight of the addition product. The addition product with a peptide having cysteinyl residue at an N-terminal showed another abnormally retarded peak in elution profile which presumably corresponded to a cyclic compound, a thiazane derivative. However, it was shown on the C-terminal tryptic peptide of actin (Cys Phe) that the conversion to a thiazane derivative could be avoided by hydrolyzing the succinimide portion of DACM at pH 9.0 before digestion. The R, values on paper chromatography for the addition products also depended on their molecular weights. However, the hydrophobicity of the coumarin portion of DACM and of the side chain of amino acid residues also affected the value. It was concluded that DACM was a valuable reagent for the purification and analysis of cysteine-containing peptides. I For Part II, see Ref. (2). 2 Abbreviations used: DACM, N-(7-dimethylamino-4-methylcoumarinyl) maleimide; SDS, sodium dodecyl sulfate; PME, @mercaptoethanol; ALC 2, alkali light chain 2; TN C, troponin C; TPCK, L-( I-tosylamide-2-phenyl)ethylchloromethyl ketone.