JOURNAL OF COLLOID AND INTERFACE SCIENCE 204, 104–111 (1998) ARTICLE NO. CS985568 Electrostatic and Hydrophobic Effects of Oligopeptide Insertions on Protein Adsorption Martin Malmsten, * ,1 Norman Burns,* and Andres Veide² * Institute for Surface Chemistry, S-114 86 Stockholm, Sweden; and ² Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44 Stockholm, Sweden Received December 8, 1997; accepted April 1, 1998 limited, at least compared to that of polymers and polyelec- The effects of oligopeptide insertions on the adsorption of the trolytes ( 1 – 3 ) . One of the reasons for this has been the lack protein ZZ, where Z is the IgG binding domain of staphylococcal of well-defined proteins. In early attempts, this was dealt Protein A, was investigated by in situ ellipsometry. In particular, with in a few studies by comparing the interfacial behavior the interplay between hydrophobic and electrostatic interactions of naturally occurring variants of the same protein. Examples as driving force for adsorption was investigated by studying the of such studies include that by Elbaum et al. on hemoglobin effects of oligopeptide insertions of the type T n ((AlaTrpTrp- (4) and that by Horsely et al. on lysozyme (5). Also recent Pro ) n ), N n ((AlaTrpTrpAspPro) n ), and P n ((AlaTrpTrpLysPro) n ) studies have been based on such variants (6–9). Moreover, on the adsorption at silica, methylated silica, and diaminocyclo- the occurrence of mutated variants of a given protein has hexane (DACH) plasma polymer surfaces. For comparison, the adsorption of the inserted peptide stretches was also investigated. facilitated studies of the effects of selected changes in the It was found that the adsorption of all the peptides increases with protein structure affecting, for example, the protein confor- the molecular weight at methylated silica. At silica, only the P n mational stability or the net protein hydrophobicity or peptides were found to adsorb. The net negatively charged proteins charge. Regarding the interfacial behavior of such protein modified through peptide insertions did not adsorb at the hydro- mutants, only a few proteins have been investigated system- philic and negatively charged silica, irrespective of the peptide atically so far. These include lysozyme T4 ( 10 – 13 ) , human insertion, whereas an extensive adsorption was found forthe posi- carbonic anhydrase II (14), tryptophan synthase a-subunits tively charged DACH surface for all the proteins investigated. For (15), the tryptic fragment of cytochrome b5 (16), and the hydrophobic and negatively charged methylated silica, on the IgG binding domain of staphylococcal protein A (Z) (17). other hand, the peptide insertions were found to have a major Of particular interest to the present investigation is that influence on the protein interfacial behavior, and the adsorption protein mutants could, at least in principle, be used to study followed the peptide stretch charge, thus increasing in the order ZZN n õ ZZT n õ ZZP n . These effects are discussed in terms of the effects of the protein-surface interaction on the adsorp- the relative importance of hydrophobic and electrostatic interac- tion. In particular, lysozyme T4 of different total charge has tions as driving force for the adsorption.  1998 Academic Press previously been used in this respect (11). However, despite Key Words: adsorption; amino acid; ellipsometry; peptide; rather large variations in the total charge ( /5 ° q net °/9), protein. the results previously obtained by McGuire et al. on the adsorption at silica and methylated silica were not entirely straightforward. A reason for this could be that changing the INTRODUCTION protein charge by point mutations also affected the protein structural stability ( 01.5 kcal/mol ° DDG °/0.5 kcal/ Protein adsorption is a process of importance not only in mol as compared to the wild-type protein), and, therefore, numerous biological systems but also in a range of biomedi- the effects of interactions and stability were not decoupled. cal applications. Despite this, and despite a tremendeous Another reason could be that the distribution of the charges amount of work devoted to improving our understanding of over the protein surface affects the adsorption. With the protein adsorption, much is still unknown about its mecha- former concern in mind, we previously initiated studies of nisms. For example, the understanding of the relative impor- the protein Z, where controlled variations of hydrophobicity tance of protein–protein, protein–solvent, and protein–sur- could be performed largely without affecting the protein face interactions, as well as of conformational, translational, structure or structural stability, thus effectively decoupling and counterion-entropy-related effects for adsorption is still interaction and conformational effects (17). This allowed us to obtain straightforward results regarding the effects of 1 To whom correspondence should be addressed. hydrophobic peptide stretch insertions on the adsorption of 104 0021-9797/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.