Interaction of DAPI with pepsin as a function of pH and ionic strength.
The fluorescent probe 4',6-diamidino-2-phenylindole (DAPI), extensively used with nucleic acids, has also recently been used with membranes and proteins (Favilla et al., Biophys. Chem., 46 (1993) 217-226 and Mazzini et al., Biophys. Chem. 52 (1994) 145-156, respectively). The spectroscopic changes of DAPI observed, namely a considerable enhancement of fluorescence, induced circular dichroism (CD) and absorbance spectral shifts, have been exploited to study the binding of this dye to both native and alkali denatured pepsin. Fluorescence and CD titrations show that nearly two molecules of DAPI bind to either native or alkali denatured pepsin with pH and ionic strength dependent Kd values, whereas absorbance titrations evidentiate an interaction characterized by a lower affinity and a larger number of binding sites. Therefore two kinds of interaction are proposed: a specific one, involving both hydrophobic and electrostatic forces; and a non-specific one, involving surface protein negative charges only. Finally, the behaviour of DAPI as a competitive inhibitor and the remarkable effect of pepstatin A, a specific inhibitor of pepsin, on both the CD and fluorescence spectra of DAPI in the presence of pepsin, show the involvement of the protein active site in the interaction.
Mazzini
A
A
Istituto Nazionale di Fisica della Materia (INFM), Dipartimento di Fisica, Università di Parma, Italy. mazzini@prix1.fis.unipr.it
Incerti
M
M
Favilla
R
R
D001665:Binding Sites; D002021:Buffers; D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D009994:Osmolar Concentration; D010434:Pepsin A / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D002021:Buffers; D005456:Fluorescent Dyes; D007211:Indoles; D010436:Pepstatins; D011480:Protease Inhibitors; C014195:Streptomyces pepsin inhibitor; C007293:DAPI; D010434:Pepsin A; C031375:pepstatin