Thiophosphorylation and
Fluorescent Labeling of Substrate Peptide of Protein Kinase
CHE Fa-Yun, XIA Qi-Chang*
( Shanghai Institute of Biochemistry, the Chinese Academy of Sciences,
Shanghai 200031, China )
Abstract Study on
the conditions of thiophosphorylation reaction and fluorescent labeling
reaction of the substrate of protein kinase A was carried out by using Kemptide
LRRASLG as a model. The suitable concentration of the fluorescent regent 5-{［((2-iodoacetyl)amino)ethyl］amino} naphthelene-1-sulfonic
acid (1,5-IAEDANS) and the suitable pH of labeling reaction buffer were 1.6
mmol/L and 8, respectively. Stability of the labeled peptide under the
conditions of automatic N-terminal protein sequencing, electrospray mass
spectrometry and in the presence of 0.1% trifluoroacetic acid was investigated,
respectively. The specific differences in UV spectra between the labeled and
unlabeled peptides were observed. Therefore, the possibility to detect the
thiophosphorylated and fluorophore labeled peptide during high performance
liquid chromatography peptide mapping was primarily shown.
Key Words substrate
peptide of protein kinase；
thiophosphorylation； fluorophore labeling； characterization of
labeled peptide
Received: August 6,
1999 Accepted: September 16, 1999
* Corresponding author: Tel, 86-21-64374430-278; Fax, 86-21-64338357; e-mail, [email protected]