1. An artificial membrane system was formed by spreading at air/water and oil/water interfaces, by using phosphatidylcholine and the glycoprotein fetuin (mol.wt. 48400). 2. The plot of increase of interfacial pressure against amount of protein added beneath a monomolecular film of phosphatidylcholine showed two discontinuities, corresponding to the completion of two distinct layers of protein: (a) largely denatured and closely associated with the polar head groups of phosphatidylcholine, possibly with penetration of non-polar protein groups between the phosphatidylcholine molecules and (b) an additional adsorbed layer of substantially native fetuin in either a close-packed or open-lattice array. A more compactly organized membrane was apparently formed at pH7.4 with 1mm-Mg2+ in the aqueous phase than without Mg2+; at 15mm-Mg2+, more random adsorption of protein appeared to take place. Qualitatively similar results were obtained at pH5.1 with 1mm-Mg2+. Closer initial packing of the phosphatidylcholine layer decreased both the magnitude of the interfacial pressure change and the amounts of protein bound in the two layers. 3. The amount of N-acetylneuraminic acid released by neuraminidase (EC 3.2.1.18) in the subphase was measured at pH5.1; a mean distribution of 9.7×1013 residues/cm2 was calculated for the completed second protein layer.
