Photoincorporation of azialcohol to the C1B domain of PKCdelta is buffer dependent.
Protein kinase C (PKC) is a signal transducing protein that has been implicated in binding alcohol and anesthetics. The alcohol and anesthetic binding of protein kinase C delta C1B domain has been determined previously by photolabeling and mass spectrometry [J. Das, G.H. Addona, W.S. Sandberg, S.S. Husain, T. Stehle, K.W. Miller, Identiffcation of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase C delta, J. Biol. Chem. 279 (2004) 37964-37972]. Here we studied photoincorporation of 3-azioctanol, a photoactive analog of octanol into PKC delta C1B in two buffer systems containing tris and hepes. The extent of photoincorporation was higher in hepes compared to tris as determined by high performance liquid chromatography and mass spectrometric analysis. The results are explained on the basis of the presence of number of primary hydroxyl and amino groups in tris and hepes molecules that could affect the binding of alcohol molecules to protein. This observation will be useful in selecting buffer system for biochemical studies on PKC.
Das
Joydip
J
Department of Pharmacological and Pharmaceutical Sciences, University of Houston, TX 77204, United States. jdas@uh.edu
D000777:Anesthetics / Q000737:chemistry; D001665:Binding Sites; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D006531:HEPES / Q000737:chemistry*; D000442:Octanols / Q000737:chemistry / Q000528:radiation effects*; D051745:Protein Kinase C-delta / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D014325:Tromethamine / Q000737:chemistry*
C541360:3-azioctanol; D000777:Anesthetics; D002021:Buffers; D000442:Octanols; D014325:Tromethamine; D051745:Protein Kinase C-delta; D006531:HEPES