The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue.
Hydrophobic sites on the surface of protein molecules are thought to have important functional roles. The identification of such sites can provide information about the function and mode of interaction with other cellular components. While the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, strong intrinsic quenching of Nile red and ANSA dye fluorescence is observed on binding to a cytochrome c('). Fluorescence quenching is also observed to take place in the presence of a variety of other biologically important molecules which can compromise the quantitative determination of binding constants. Absorption difference spectroscopy is shown not to be sensitive to the presence of fluorescence quenchers but sensitive enough to measure binding constants. The dye BPB is shown to bind to the same hydrophobic sites on proteins as polarity-sensitive fluorescence probes. The absorption spectrum of BPB is also observed to be polarity sensitive. A binding constant of 3x10(6)M(-1) for BPB to BSA has been measured by absorption difference spectroscopy. An empirical correlation is observed between the shape of the absorption difference spectrum of BPB and the polarity of the environment. The results indicate that absorption difference spectroscopy of BPB provides a valuable supplement to fluorescence for determining the presence of hydrophobic sites on the surface of proteins as well as a method for measuring binding constants.
Bertsch
M
M
Department of Chemistry, University of Illinois at Chicago, Science and Engineering South 845 West Taylor Street, Rm. 4500, Chicago, IL 60607, USA.
Mayburd
A L
AL
Kassner
R J
RJ
D001665:Binding Sites; D001667:Binding, Competitive; D001978:Bromphenol Blue / Q000737:chemistry* / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005470:Fluorometry / Q000379:methods; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods
D003574:Cytochrome c Group; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D001978:Bromphenol Blue; D012710:Serum Albumin, Bovine