Direct Measurements of the Interaction between Layers of Insulin Adsorbed on Hydrophobic Surfaces PER M. CLAESSON,*'t 'l THOMAS ARNEBRANT,~ BJORN BERGENSTAHL,* AND TOMMY NYLANDER:~ *Department of Physical Chemistry, The Royal Institute of Technology, S-100.44 Stockholm, Sweden, and The Institute for Surface Chemistry, Box 5607, S-114.86 Stockholm, Sweden; t Department of Applied Mathematics, Research School of Physical Sciences, Institute of Advanced Studies, The Australian National University, GPO Box 4, ACT 2600, Australia; and ~Department of Food Technology, University of Lund, Box 124, S-221.00 Lund, Sweden Received May 16, 1988; accepted August 16, 1988 The forces acting between layers of insulin adsorbed on mica surfaces rendered hydrophobic by de- position of dioctadecyldimethylammonium bromide have been investigated. The forces have been de- termined after different adsorption times, and after dilution at different desorption times and pH values. The adsorption of insulin has a dramatic effect on the surface forces. The long-range attraction, which is present when no insulin molecules are adsorbed, disappears. Instead a repulsive double-layer force dominates the long-range interaction. An attractive force was in some cases observed at intermediate distances (6-12 nm). The actual value of this force was dependent on how fast the measurements were carried out, indicating that molecules in the interlayer region rearranged or were pushed out from between the surfaces slowly. At small separations a repulsive force caused by steric and dehydration effects pre- dominates. The upper limit of the range of the hydration force between insulin coated surfaces is about 1 Bin. © 1989 Academic Press, Inc. INTRODUCTION The various amino acid side chains of pro- teins interact with each other strongly through disulfide bridges, hydrogen bonds, dipole, and hydrophobic interactions. These strong and versatile internal interactions are the reason that most proteins are folded and have a re- stricted flexibility. Hence, in some respects the solution properties of many proteins are more reminiscent of colloidal sols than of solutions of flexible polymers. Fundamental questions, such as the solution stability, gel formation, surface activity, and a more general function- ality are directly related to interactions occur- ring at the protein-water interface. These in- teractions involve nonspecific forces such as double-layer forces, hydration forces, hydro- phobic forces, and van der Waals forces. But To whom correspondence should be addressed. they also involve specific, directional depen- dent forces like hydrogen bonds, acid-base in- teractions, and dipole-dipole interactions op- erating on an atomic scale. Further, in, e.g., enzyme substrate reactions, covalent bonds are broken up and new bonds may be formed. Despite the enormous interest in biopoly- mers, only a few studies of interactions in such systems have been carried out. Among those are investigations of the interaction between layers of mucin ( 1), collagen (2), and myelin basic protein (3) adsorbed on mica. Further, Rau and co-workers determined the forces acting between condensed DNA molecules by applying an osmotic stress method (4). They found a strong repulsive force at separations between 0.5 and 1.5 nm which was indepen- dent of the electrolyte concentration. This re- pulsion is presumably of an origin similar to that of the hydration forces acting between phospholipid bilayers (5-7). 457 Journal of Colloid and Interface Science, Vol. 130, No. 2, July 1989 0021-9797/89 $3.00 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.