title: Modulation of lysozyme charge influences interaction with phospholipid vesicles.
abstract: Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between charge state of the protein and its interaction with negatively charged phospholipid membranes chemical modifications of the proteins were carried out. Succinylation and carbodiimide modification was used to shift the isoelectric point of lysozyme to lower and higher pH values, respectively. The binding of the modified lysozyme to phospholipid vesicles prepared from phosphatidic acid (PA) was determined using microelectrophoresis and ultracentrifugation. At acidic pH of the solution all lysozyme species reduced the surface charges of PA vesicles. Succinylated lysozyme (succ lysozyme) reduced the electrophoretic mobility (EPM) to nearly zero, whereas native lysozyme and carboxylated lysozyme (carbo lysozyme) changed the surface charge to positive values. At neutral pH, the reduction of surface charges was less for carbo lysozyme and unmodified lysozyme. Succ lysozyme did not change the EPM. Unmodified and carbo lysozyme decreased the magnitude of EPM, but the whole complex was still negatively charged. The bound fraction of all modified lysozyme to PA vesicles at high lysozyme/PA ratios was nearly constant at acidic pH. At low lysozyme/PA ratios the extent of bound lysozyme is changed in the order carbo>unmodified>succ lysozyme. Increasing the pH, the extent of bound lysozyme to PA large unilamellar vesicles (LUV) is reduced, at pH 9.0 only 35% of carbo lysozyme, 23% of unmodified lysozyme is bound, whereas succ lysozyme does not bind at pH 7.4 and 9.0. At low pH, addition of all lysozyme species resulted in a massive aggregation of PA liposomes, at neutral pH aggregation occurs at much higher lysozyme/PA ratios. Lysozyme binding to PA vesicles is accompanied by the penetration of lysozyme into the phospholipid membrane as measured by monolayer techniques. The penetration of lysozyme into the monolayer was modulated by pH and ionic strengths. The interaction of lysozyme with negatively charged vesicles leads to a decrease of the phospholipid vesicle surface hydration as measured by the shift of the maximum of the fluorescence signal of a headgroup labeled phospholipid. The binding of bis-ANS as an additional indicator for the change of surface hydrophobicity is increased at low pH after addition of lysozyme to the vesicles. More hydrophobic patches of the lysozyme-PA complex are exposed at low pH. At low pH the binding process of lysozyme to PA vesicles is followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the vesicle aqueous content. The extent of lysozyme interaction with PA LUV at neutral and acidic pH is in the order carbo lysozyme>lysozyme>succ lysozyme.
authors.affiliation: University of Leipzig, Institute for Medical Physics and Biophysics, Härtelstrasse 16-18, D-04107 Leipzig, Germany. zsco@medizin.uni-leipzig.de
authors.forename: Olaf
authors.initials: O
authors.lastname: Zschörnig
authors.forename: Gerrit
authors.initials: G
authors.lastname: Paasche
authors.forename: Cathrin
authors.initials: C
authors.lastname: Thieme
authors.forename: Nikola
authors.initials: N
authors.lastname: Korb
authors.forename: Klaus
authors.initials: K
authors.lastname: Arnold
mesh_terms: D001665:Binding Sites; D002234:Carbodiimides / Q000737:chemistry / Q000378:metabolism*; D004586:Electrophoresis; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008081:Liposomes / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D010712:Phosphatidic Acids / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013386:Succinates / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D014461:Ultracentrifugation
chemical_list: D002234:Carbodiimides; D008081:Liposomes; D010712:Phosphatidic Acids; D010743:Phospholipids; D013386:Succinates; C110021:N-succinyl lysozyme; D009113:Muramidase