Proteins at Interfaces III State of the Art 9780841227965, 9780841227972

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Proteins at Interfaces III State of the Art 2012

In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Downloaded by FORDHAM UNIV on December 13, 2012 | http://pubs.acs.org Publication Date (Web): December 12, 2012 | doi: 10.1021/bk-2012-1120.fw001

ACS SYMPOSIUM SERIES 1120

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Proteins at Interfaces III State of the Art 2012 Thomas Horbett, Editor University of Washington Seattle, Washington

John L. Brash, Editor McMaster University Hamilton, Ontario, Canada

Willem Norde, Editor Wageningen University Wageningen, The Netherlands and

University Medical Center Groningen and University of Groningen Groningen, The Netherlands Sponsored by the ACS Division of Colloid and Surface Chemistry

American Chemical Society, Washington, DC Distributed in print by Oxford University Press, Inc.

In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Library of Congress Cataloging-in-Publication Data

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Library of Congress Cataloging-in-Publication Data Proteins at interfaces III : state of the art 2012 / Thomas Horbett, editor, University of Washington, Seattle, Washington ; John L. Brash, editor, McMaster University, Hamilton, Ontario ; Willem Norde, editor, Wageningen University, Wageningen, The Netherlands and University Medical Center Groningen and University of Groningen, Groningen, The Netherlands ; sponsored by the ACS Division of Colloid and Surface Chemistry. pages cm. -- (ACS symposium series, ISSN 0097-6156 ; 1120) Includes bibliographical references and index. ISBN 978-0-8412-2796-5 (alk. paper) 1. Proteins--Congresses. 2. Surface chemistry--Congresses. 3. Biological interfaces-Congresses. I. Horbett, Thomas A., 1943- editor of compilation. II. American Chemical Society. Division of Colloid and Surface Chemistry. III. Title: Proteins at interfaces three. IV. Title: Proteins at interfaces 3. QP551.P6977822 2012 612′.01575--dc23 2012043084

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48n1984. Copyright © 2012 American Chemical Society Distributed in print by Oxford University Press, Inc. All Rights Reserved. Reprographic copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Act is allowed for internal use only, provided that a per-chapter fee of $40.25 plus $0.75 per page is paid to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. Republication or reproduction for sale of pages in this book is permitted only under license from ACS. Direct these and other permission requests to ACS Copyright Office, Publications Division, 1155 16th Street, N.W., Washington, DC 20036. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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Foreword The ACS Symposium Series was first published in 1974 to provide a mechanism for publishing symposia quickly in book form. The purpose of the series is to publish timely, comprehensive books developed from the ACS sponsored symposia based on current scientific research. Occasionally, books are developed from symposia sponsored by other organizations when the topic is of keen interest to the chemistry audience. Before agreeing to publish a book, the proposed table of contents is reviewed for appropriate and comprehensive coverage and for interest to the audience. Some papers may be excluded to better focus the book; others may be added to provide comprehensiveness. When appropriate, overview or introductory chapters are added. Drafts of chapters are peer-reviewed prior to final acceptance or rejection, and manuscripts are prepared in camera-ready format. As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproductions of previous published papers are not accepted.

ACS Books Department

In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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Preface This book is based on the Proteins at Interfaces III symposium held at the 243rd American Chemical Society meeting in San Diego March of 2012. The symposium was sponsored by the Colloid and Surface Science division, whose support and help we gratefully acknowledge, especially the warm welcome and continuous aid given to us by the program chairman Ramanathan Nagarajan. We also wish to thank the American Chemical Society book division for agreeing to publish this volume, in particular our editor Timothy Marney who helped us throughout to keep on time with the many tasks involved in preparing this volume. Ms. Arlene Furman was our invaluable assistant editor throughout the process, and we are especially grateful for her thorough work in guiding us through the many steps involved in getting each manuscript through the review process. The authors who contributed chapters are thanked again for their willingness to share their knowledge of Proteins at Interfaces. Finally, we wish to acknowledge the contribution of Dr. Dan Li, Soochow University, who provided the cover art. The chapters in the book are grouped into five general areas: physical chemistry, computer simulation, biological effects, protein resistant surfaces, and techniques for the study of protein adsorption and adsorbed proteins. We considered these to be major categories into which the research in this area falls; the introductory chapter is organized along the same lines. We also wish to point out that the various chapters typically include elements that represent more than one of these areas; we placed them in the topic area we felt they were most closely related to. As the book’s title indicates, this is the third volume of its type to appear. The prior two also originated from symposia sponsored by the ACS Colloid and Surface Science division, and were published by ACS books division: ACS Symposium Series Vol. 343, Proteins at Interfaces, Physicochemical and Biochemical Studies, J. L. Brash and T. A. Horbett, editors, American Chemical Society, Washington, D.C., 1987; ACS Symposium Series Vol. 602, Proteins at Interfaces II: Fundamentals and Applications, T. A. Horbett and J. L. Brash, editors, ACS Books, Washington, D.C., 1995. The general intent for all three initiatives was the same, namely to bring together the many groups around the world working on proteins at interfaces to share their ideas and knowledge, and to document the current state of the art in the resulting publication. Thomas A. Horbett Departments of Bioengineering and Chemical Engineering University of Washington Seattle, Washington 98195 xi In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

John L. Brash School of Biomedical Engineering, Department of Chemical Engineering McMaster University Hamilton, Ontario, Canada L8S 4L8

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Willem Norde Department of Biomedical Engineering University Medical Center Groningen Groningen, The Netherlands Laboratory of Physical Chemistry and Colloid Science Wageningen University The Netherlands

xii In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Editors’ Biographies

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Thomas A. Horbett Thomas A. Horbett (Ph.D., University of Washington) is currently a professor emeritus in Bioengineering and Chemical Engineering at the University of Washington in Seattle, where he spent his entire career doing research on the interactions of biomaterials with blood and soft tissue. His research was supported for many years by grants from the National Institute of Health. Specific research areas he has worked in include protein adsorption, cell interactions, blood compatibility, the foreign body reaction, and controlled delivery of insulin and other drugs from polymers. He is the recipient of the 1989 Society for Biomaterials Clemson Award for Basic Research. He was the Distinguished Lecturer in Controlled Drug-Delivery,1994 College of Pharmacy, Rutgers University, and is a Fellow, Society for Biomaterials (1994); Fellow, American Institute of Medical and Biological Engineering (1995); and Fellow, Biomaterials Science Engineering (FBSE) of the World Biomaterials Congress (1996). He has authored or co-authored 125 journal articles, and co-edited 3 books, including Proteins at Interfaces I and II. He has also organized or co-organized 5 symposia at national meetings since 1975. He is a long-standing member of the editorial board of the Journal of Biomedical Materials Research, the Journal of Biomaterials Science, Polymer Edition, and the Journal of Colloids and Surfaces B: Biointerfaces.

John L. Brash John Brash is Distinguished University Professor, McMaster University, in the Department of Chemical Engineering and the School of Biomedical Engineering. He has worked in biomaterials and biocompatibility research for most of his career, with emphasis on materials for use in blood contact, which are required for devices such as vascular grafts, coronary stents, and heart valves. He is the author of over 200 peer-reviewed journal publications, most of which are in the biomaterials area with a strong focus on protein–surface interactions. His work has involved collaborations with groups in Canada, the United States, Germany, France, Sweden, Australia, and China. Honors include: Fellow, Royal Society of Canada (FRSC); Fellow, Biomaterials Science and Engineering (FBSE), International Union of Societies for Biomaterials Science and Engineering; Clemson Award for Basic Research, U.S. Society for Biomaterials; Founders Award, U.S. Society for Biomaterials; Chair Professor, Soochow University, Suzhou, China; R.S. Jane Memorial Award, Canadian Society for Chemical Engineering; Docteur Honoris Causa, Université de Paris (XIII); and Chair, © 2012 American Chemical Society In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Gordon Research Conference on Biocompatibility and Biomaterials. He is a present or past member of several editorial boards, including those of the Journal of Biomedical Materials Research, Biomaterials, Journal of Biomaterials Science Polymer Edition, Journal of Colloid and Interface Science, and Biointerphases. He is currently an editor of Colloids and Surfaces B: Biointerfaces.

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Willem Norde Willem Norde (Ph.D., Wageningen University, The Netherlands) is professor emeritus in Bionanotechnology at Wageningen University and professor in Colloid and Interface Science at the University Medical Center Groningen and the University of Groningen, The Netherlands. He has a long and internationally acknowledged expertise in studying interactions among different types of (bio)particles, in particular proteins and micro-organisms, and between such particles and surfaces. He has (co-)authored about 250 peer-reviewed publications; his H-index is 55. He wrote several book chapters, edited Physical Chemistry of Biological Interfaces (Marcel Dekker, 2000), Nanotechnology in the Agri-Food Sector (Wiley-VCH, 2011) and is author of the book Colloids and Interfaces in Life Sciences and Bionanotechnology (CRC Press, 2011). He is member of the editorial board of Colloids and Surfaces B: Biointerfaces, and a member of the International Association of Colloid and Interface Scientists, the European Colloid and Interface Society, the Royal Dutch Chemical Society, and the Dutch Society for Biotechnology. He has been visiting professor at various universities around the world.

858 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Chapter 1

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Willem Norde,1,2 Thomas A. Horbett,3 and John L. Brash*,4 1Department

of Biomedical Engineering, University Medical Center Groningen, Groningen, The Netherlands 2Laboratory of Physical Chemistry and Colloid Science, Wageningen University, The Netherlands 3Departments of Bioengineering and Chemical Engineering, University of Washington, Seattle, Washington 98195 4School of Biomedical Engineering, Department of Chemical Engineering, McMaster University, Hamilton, Ontario, Canada L8S 4L8 *E-mail: [email protected]

In this chapter, we provide a review of current research on proteins at interfaces under the headings: physicochemical aspects, computer simulation of protein adsorption, biological function of adsorbed proteins, resistance to protein adsorption, and experimental techniques for the study of protein surface interactions. All of these areas are represented in the various chapters in the book. This chapter gives a broader context into which the individual, specialized chapters can be placed and we have attempted to point out the connections. We intend this chapter to be of help to the community at large, and in particular to beginning students and new investigators wishing to make a contribution to the field.

1. Physicochemical Aspects Proteins are very complex polymers. They are polyamino acids built from twenty two different amino acids, linked together via peptide bonds. They vary in size, polarity and charge. Depending on the distribution of the polar and apolar amino acids along the polyamino acid chain, the protein molecule is more or less amphiphilic. This is one of the more general or overriding reasons why © 2012 American Chemical Society In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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proteins are very surface active. Furthermore, some amino acids in the polymer chain contain a cationic group, some an anionic group, and others are uncharged. Proteins may therefore be classified as polyampholytes. Just as an almost infinite number of words can be written by using the twenty six letters of the alphabet, an endless number of polyamino acids may be formed using the twenty two amino acids, each one with its own amino acid composition and distribution. As with words, only a fraction (but still amounting to millions) of all possible sequences are “meaningful”, that is, are represented in nature as proteins, each with its own specific function. Understanding the behavior of proteins at interfaces may start from that of the simple, coiled polymers. First, like the simple polymers, proteins adsorb by attaching several segments to a surface (like a centipede on a fly trap) resulting in a poor ability to desorb (1–3). When the affinity for attachment of the various molecular segments is sufficiently reduced by environmental changes (e.g., temperature, pH, ionic strength, etcetera), the protein may leave the surface. Also, protein molecules may be displaced from the surface by adding components that have a higher affinity to adsorb. Second, because of their ionic groups proteins show adsorption patterns typical for polyampholytes, that is, strong pH-dependence, the more so the lower the ionic strength, with a maximum adsorbed amount at isoelectric conditions (4). In other aspects the adsorption behavior of most proteins deviates from that of the simple polymers. In solution the simple polymers adopt flexible high-entropy structures, but when adsorbed at an interface their entropy is lower. In proteins, in particular globular proteins in an aqueous medium, the polyamino acid chain is folded up to shield the apolar moieties from contact with water resulting in a more or less compact structure of which the exterior is relatively hydrophilic and the interior more hydrophobic. Obviously, the ionic groups reside primarily at the water-exposed surface of the protein molecule. Thus, unlike the simple polymers, proteins have limited conformational freedom or, in other words, are low-entropy structures (5). For reasons explained under “Protein adsorption affinity”, upon adsorption the protein may undergo structural rearrangements towards a higher conformational entropy. Against this background we will discuss some theoretical and phenomenological aspects of protein adsorption and its applications. 1.1. Protein Adsorption Affinity Adsorption data are often presented in the form of adsorption isotherms, where, for constant temperature, the adsorbed amount Γ per unit mass or, preferably, per unit surface area of the sorbent, is plotted against the protein concentration cp in solution, after adsorption. Protein adsorption isotherms tend to belong to the “high affinity” category, displaying a steep initial rise and a strong resistance to desorption by dilution. There is no reason to expect the isotherms to be of the Langmuirian type, because the premises of the Langmuir theory are usually not fulfilled: the adsorption is not at all or only partly reversible, lateral interactions cannot be excluded, and the attachment is usually not site-determined (6). The observation that adsorption isotherms for (globular) proteins show 2 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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well-developed plateau values, unlike the isotherms for coiled polymers that tend to increase with increasing concentration in solution, suggests that, even though structural changes may occur, the protein molecules do not unfold to attain a loopy structure at the sorbent surface. Indeed, different experimental approaches, such as ellipsometry, optical waveguide spectroscopy, quartz crystal microbalance, and AFM spectroscopy, point to relatively compact adsorbed protein layers (7–10). In some cases protein adsorption isotherms do follow a Langmuir pattern. This may be due to variation of the sorbent surface area occupied per protein molecule with varying protein concentration in solution. This phenomenon is discussed in more detail in section “Kinetics and dynamics of protein adsorption” as well as in reference (11). Obviously, analysis of the isotherm using the Langmuir theory (or modifications thereof) yields misleading conclusions, because the underlying conditions of that theory are not obeyed. Because of their complex nature, i.e., amphiphilicity, ambivalency, and structural features, adsorption of proteins is an intricate phenomenon involving different types of interactions. The main contributions to the adsorption process are from electrostatic, dispersion and hydrophobic forces, and, in many cases, from rearrangements in the structure of the protein molecules (6). The distance over which electrostatic interaction is effective, the so-called Debye length (12), is in the range of a few nm, depending on the ionic strength. More specifically, in a medium of 0.01 M ionic strength the Debye length is 3 nm and in 0.1 M ionic strength it is 1 nm. Dispersion forces between proteins and sorbents interacting across an aqueous medium are usually attractive but small, because of the small dimensions of protein molecules and the low value of the Hamaker constant pertaining to such systems (12, 13). When the protein and the sorbent are in close proximity (say, ≤ 0.5 nm) changes in the hydration of both components may strongly affect the adsorption. When the surfaces of the protein and the sorbent are both polar it is probable that some hydration water is retained in the contact zone between the two. However, if the surface of the protein and/or the sorbent is primarily apolar, dehydration strongly favors adsorption. Thus, when the protein and the sorbent repel each other electrostatically adsorption may occur because of overruling attractive forces. More quantitatively, the Gibbs energy of dehydration of one CH2 group is about 1 kBT, which corresponds to the Gibbs energy of adsorption of one monovalent ion at a surface having a potential of 25 mV. Still, because of the larger range of operation, electrostatic repulsion may give rise to an energy barrier that the protein has to surpass prior to deposition at the sorbent surface. Rearrangements in the protein structure may occur when a protein molecule encounters an interface where it can turn one side away from the aqueous solution. Then, upon adsorption the protein may be able to present part of its hydrophobic interior at the sorbent surface without exposing apolar residues to the water. As a consequence, intramolecular hydrophobic interactions become less important as a factor stabilizing the protein structure. Because hydrophobic interactions in the protein’s interior support the formation of ordered secondary structures (α-helices and β-sheets) (5), a reduction of these interactions destabilizes such structures. A decrease of α-helix and/or β-sheet content is therefore expected if the peptide units released from these structures can form hydrogen bonds with the 3 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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sorbent surface, as is the case for oxides like glass, silica, and metal oxides, or with remaining surface-bound water molecules. The decrease of ordered structure implies a higher conformational entropy and thus favors adsorption, possibly up to tens of kBT per protein molecule (6). If, however, in the non-aqueous contact zone no hydrogen bonding with the surface is possible, which is the case for apolar surfaces, adsorption may induce extra peptide-peptide hydrogen bonds promoting the formation of α-helices and β-sheets (14). Thus, whether or not adsorption at an apolar surface leads to an increased or decreased order in the protein structure depends on a subtle balance between energetically favorable intramolecular interactions (notably hydrogen bonding) and the ensuing changes in the conformational entropy of the protein. In this context the terms “hard” and “soft” have been introduced (15) to indicate the strength of the internal structural coherence in the protein molecule and, hence, its resistance against adsorption-induced conformational changes. The main conclusion is that interfaces cannot easily resist the adsorption of proteins. When the sorbent surface is hydrophobic, adsorption of any type of protein is very likely because dehydration of that surface easily outweighs electrostatic repulsion. When the sorbent is hydrophilic electrostatic interaction and/or protein structural changes may facilitate adsorption. Only when the surface is hydrophilic and the protein hard can electrostatic repulsion prevent adsorption from occurring. To achieve protein resistance, surfaces are modified, e.g., by applying a coating of hydrophilic strongly hydrated polymers or zwitterionic components. This matter is further discussed in Section 4, Resistance to protein adsorption.

1.2. Kinetics and Dynamics of Protein Adsorption As adsorption of proteins appears to be irreversible on practical time scales, the characteristics of the adsorbed molecules in their final state depend on their history, that is, on their preceding stages. Kinetics, in particular rates of adsorption relative to rates of structural changes, should be considered. During the last few decades various models for protein adsorption kinetics have been proposed. Because of the complexity of the protein and, possibly, the sorbent surfaces on the atomic level the models follow a mesoscopic approach, where the protein is considered as a particle and effective rate constants, particle-sorbent and particle-particle interactions are used. The models have in common that they account for the generally observed features of (partial) irreversibility of the protein adsorption process and deceleration of adsorption with increasing coverage of the sorbent surface. The models differ more or less with respect to the underlying assumptions. For instance, Bornzin and Miller (16) assume the sorbent surface heterogeneity to cause partial irreversibility, distinguishing regions where the protein molecules stick irreversibly and regions where they attach weakly and desorb upon dilution. Kurrat et al (17) interpret reversible and irreversible binding in terms of the number of bonds formed, without indicating whether this variation in the way of binding results from sorbent surface heterogeneity or from different orientations/conformations of the 4 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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adsorbed protein molecule. Obviously, when the sorbent surface is homogeneous the irreversibly adsorbed fraction will increase in time and ultimately the whole protein population at the surface will be irreversibly adsorbed. In the models proposed by Walton and Soderquist (18) and Beissinger and Leonard (19) time-dependent adaptation of the adsorbed protein structure to optimize interaction with the sorbent surface is accounted for: initially the protein adsorbs reversibly but during contact with the surface the desorption rate decreases gradually with time. Here too, the irreversible nature of the adsorbed layer increases with ongoing contact between the sorbent and the protein solution. The same assumptions, but, furthermore, an increasing molecular area (“footprint”) of the adsorbed protein molecule when it relaxes at the sorbent surface, is included in the model presented by Norde (20). According to this model, the growing fraction of irreversibly adsorbed, structurally relaxed, molecules at the expense of reversibly adsorbed unperturbed ones may result in an “overshoot” of protein at the surface during the course of the adsorption process (21–23). Perhaps the most successful, at least the most popular, description of protein adsorption is the random sequential adsorption (RSA) model, or modifications thereof (24–26). According to the RSA theory a single adsorbed molecule (or, for that matter, particle) that hits the sorbent surface sticks there and defines a zone around that particle that excludes the center of subsequently arriving molecules. Thus, for spheres of radius a each adsorbing particle blocks an area of π(2a)2. Therefore, at low surface coverage the area available for adsorption decreases four times faster than when the surface occupancy by the particles themselves is taken into account, with a corresponding decrease in adsorption rate. At higher surface coverage the area available for adsorption should be corrected for overlapping exclusion zones around the particles. For spheres the RSA model predicts adsorption saturation due to jamming at a surface coverage of 55%. For particle geometries deviating from spherical the jamming limit is lower, e.g., 40% for particles having an aspect ratio of 7.5. Experimental values for protein adsorption are usually higher than the jamming limits predicted by RSA. This may be due to the possibility of lateral diffusion of the adsorbed protein molecules, as has been reported by (27, 28) as well as by Sotres et al in Chapter 6. A fundamental problem in applying this or any model to actual data is that the area available to the protein molecule is never well known, because even very small, protein molecule sized deviations from flatness can accommodate protein molecules, yet are easily missed by surface area measuring methods. The RSA model may be modified to more accurately describe protein adsorption by including surface-induced changes in protein conformation and orientation (26). Such changes usually lead to a larger footprint and in the RSA model this is accounted for by an instantaneous and symmetric expansion of the particle to a given pre-set size. If no space is available for that expansion the particle permanently keeps its original dimensions. Further modification includes that, unlike the expanded particle, the non-expanded particles may leave the surface by desorption (26). The result could be that, under certain conditions, non-expanded particles are gradually displaced by expanded ones showing up as a maximum in the adsorbed amount (“overshoot”) during the sequential adsorption process. 5 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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The RSA model has also been adapted to apply to mixtures of different proteins (26). Then, the population of each type of protein in the adsorbed layer is determined by its respective adsorption rate and molecular size. However, the outcome is not compatible with the experimentally observed “Vroman effect”, i.e., the transient change in composition of the adsorbed layer due to the displacement one kind of protein by a later arriving kind that has a higher adsorption affinity (29, 30). Indeed, the experimental data on sequential adsorption of antibodies and serum albumin, reported by Dupont-Gillain in Chapter 21, seem to be at odds with the RSA model. Each of these models is suspect in one way or another. The main problem is related to the complexity and heterogeneity of the protein molecule and the poor understanding of the mechanism underlying the time-dependent desorbability of the protein layer (31). Structural changes in the protein molecules are essential in adsorption kinetics and, because of the irreversibility of the adsorption process, for the final state and, as a consequence, for the biological activity of the adsorbed layer. Therefore, the dynamics of the relaxation of the proteins at the sorbent surface is considered in more detail. Relaxation, which usually implies a certain degree of spreading, leading to lower adsorption saturation Γsat, occurs with a certain characteristic time τr. The extent of spreading depends on the rate of relaxation compared to the time τf needed to fill the sorbent surface, in the absence of desorption. The value of τr depends on the protein’s resistance against deformation. For a given protein, the internal coherence usually decreases with increasing net charge. Indeed, the maximum value of Γsat (pH), often observed at the isoelectric point of the protein, may be ascribed to progressive conformational changes at pH values further away from the isoelectric point (6). Additionally, τr is influenced by properties of the sorbent-water interface, notably its interfacial tension. The higher the interfacial tension is, the stronger is the tendency to spread over it. Examples are given in references (32, 33). The value of τf is controlled by the supply rate (flux) of molecules that arrive at the sorbent surface and are able to deposit. Hence, τf scales inversely with the protein concentration in solution and linearly with the resistance to reach the surface (which is composed of the resistance to transport through the solution and the resistance associated with overcoming possible barriers for deposition). If τr/τf >>1, relaxation is completely inhibited because adjacent surface area is already occupied by newly depositing molecules before the previously adsorbed one has the time to spread. If, conversely, τr/τf