THE APPLICATION OF PH-SENSITIVE SPIN LABELS TO STUDIES OF SURFACE-POTENTIAL AND POLARITY OF PHOSPHOLIPID-MEMBRANES AND PROTEINS

The effects of pH titration on the EPR spectra of imidazolidine nitroxides located at the surface of mixed bilayers composed of dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylcholine (DMPC), and at the surface of the protein, human serum albumin (HSA), have been investigated. It is found that the shift in pK(a) of the amino group of the imidazolidine radical from its value of 4.6 in water depends both on the interfacial polarity (DELTA-pK(a)pol) and on the electrostatic surface potential (DELTA-pK(a)el) when it is positioned at the bilayer/water interface by an anchoring hydrocarbon tail. The polarity shift is determined to be: DELTA-pK(a)pol = -1.3 units at the surface of DMPC bilayers at 17-degrees-C, corresponding to an effective interfacial dielectric constant of epsilon almost-equal-to 37, and depends on the temperature with a coefficient of d DELTA-pK(a)pol/dT almost-equal-to -0.01 per degree. The electrostatic shift at the surface of DMPG bilayers is DELTA-pK(a)el = +1.6 units in 0.1 M KCl, which corresponds to an electrostatic surface potential of -95 mV. This electrostatic shift depends strongly both on ionic strength and on the fraction of charged lipid in the DMPC/DMPG mixtures, in a manner that agrees with the predictions of electrostatic double-layer theory. It is found that the shift in pK(a) of an imidazolidine radical covalently bound at the surface of HSA is determined mainly by the surface electrostatics (DELTA-pK(a)pol almost-equal-to 0) and corresponds to an electrostatic potential of +33 mV in 0.01 M KCl at a pH below the isoelectric point of the protein.