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This thesis shows the effects that the addition of low molecular weight surfactants (LWMS) to proteins has on the foam stability of the mixture. For this, the bulk, interfacial, thin liquid films and foam properties are determined for different protein-LWMS mixtures at different molar ratios (MR). It was shown that the MR as well as the charge of the protein and LMWS determine the foam stability of the mixtures. For all mixtures it was found that the proteins have a select number of high affinity binding sites. So, the concentration of free LMWS in the solution is 0 until a critical MR (MRcr), at which all high affinity binding sites are saturated. Above this MRcr, part of the LMWS binds to low affinity binding sites of the proteins. The low affinity binding sites have a binding ratio < 1, which determines the concentration of bound and free LMWS. For similarly charged protein-LMWS mixtures (i.e. b-lactoglobulin (BLG) and sodium dodecyl sulphate (SDS) and bovine serum albumin (BSA) and SDS at pH 7) the foam stability typically decreases from the foam stability of the pure protein solution (MR 0) until MRcr is reached. At MR > MRcr the foam stability is dominated by the amount of free LMWS. For oppositely charged protein-LMWS mixtures, the binding of the LMWS to the proteins can be described in a similar way, although the number of high affinity sites and low affinity binding ratio are different. There is also a regime of MRs in which the protein-LMWS complexes form large aggregates. These aggregates were in some cases found to increase foam stability (lysozyme (LYS) and SDS and BLG-SDS at pH 3), while in another case (BLG and cetyltrimethylammonium bromide (CTAB)) they lead to decreased foam stability. Still, in all cases it was found that above MRD the aggregates dissociate and the foam stability becomes dominated by free surfactants, equivalent to what was observed for similarly charged protein-LMWS mixtures. A multi-scale model was developed to describe the stability of thin liquid films in terms of rupture time and thickness. Initially, the model was used to predict the stability of surfactant free films of water and electrolyte solutions. Later, it was used to predict the foam stability in LYS-SDS mixtures. For that purpose, the model was combined with a foam drainage model to provide theoretical estimations of foam stability. This model is the basis to understand coalescence of bubbles in foam. Finally, the concept of the critical MRs and the free LMWS was introduced. Using this, the foam properties of protein-LMWS mixtures can partly be predicted by relative charge of the components and the binding to both high and low affinity binding sites.

This thesis shows the effects that the addition of low molecular weight surfactants (LWMS) to proteins has on the foam stability of the mixture. For this, the bulk, interfacial, thin liquid films and foam properties are determined for different protein-LWMS mixtures at different molar ratios (MR). It was shown that the MR as well as the charge of the protein and LMWS determine the foam stability of the mixtures. For all mixtures it was found that the proteins have a select number of high affinity binding sites. So, the concentration of free LMWS in the solution is 0 until a critical MR (MRcr), at which all high affinity binding sites are saturated. Above this MRcr, part of the LMWS binds to low affinity binding sites of the proteins. The low affinity binding sites have a binding ratio < 1, which determines the concentration of bound and free LMWS. For similarly charged protein-LMWS mixtures (i.e. b-lactoglobulin (BLG) and sodium dodecyl sulphate (SDS) and bovine serum albumin (BSA) and SDS at pH 7) the foam stability typically decreases from the foam stability of the pure protein solution (MR 0) until MRcr is reached. At MR > MRcr the foam stability is dominated by the amount of free LMWS. For oppositely charged protein-LMWS mixtures, the binding of the LMWS to the proteins can be described in a similar way, although the number of high affinity sites and low affinity binding ratio are different. There is also a regime of MRs in which the protein-LMWS complexes form large aggregates. These aggregates were in some cases found to increase foam stability (lysozyme (LYS) and SDS and BLG-SDS at pH 3), while in another case (BLG and cetyltrimethylammonium bromide (CTAB)) they lead to decreased foam stability. Still, in all cases it was found that above MRD the aggregates dissociate and the foam stability becomes dominated by free surfactants, equivalent to what was observed for similarly charged protein-LMWS mixtures. A multi-scale model was developed to describe the stability of thin liquid films in terms of rupture time and thickness. Initially, the model was used to predict the stability of surfactant free films of water and electrolyte solutions. Later, it was used to predict the foam stability in LYS-SDS mixtures. For that purpose, the model was combined with a foam drainage model to provide theoretical estimations of foam stability. This model is the basis to understand coalescence of bubbles in foam. Finally, the concept of the critical MRs and the free LMWS was introduced. Using this, the foam properties of protein-LMWS mixtures can partly be predicted by relative charge of the components and the binding to both high and low affinity binding sites.

Proteine, umgangssprachlich auch Eiweiße genannt, bestehen aus einer linearen Polypeptidkette aus bis zu 20 verschiedenen Aminosäuren. Sie sind nahezu an allen biologischen Prozessen beteiligt und bilden die Grundbausteinen aller Zellen. Ihre letztendlich spezielle funktionelle Eigenschaft resultiert aus ihrer dreidimensionalen Form. Steht eine Proteinlösung in Kontakt mit einer festen Oberfläche oder einer Oberfläche mit fluidem Charakter (Polyelektrolyt-Bürsten), adsorbieren Proteine spontan an der Flüssigkeit/Festkörper-Grenzfläche. Eine große Anzahl von Triebkräften wird für diesen Vorgang diskutiert. Hierzu zählen u. a. Konformationsänderungen des Proteins, elektrostatische Anziehungskräfte, van der Waals-Kräfte und das Modell der counterion release force. Zur Untersuchung der Adsorption der Proteine Hühnereiweiß-Lysozym, Staphylokokken-Nuclease (SNase), sowie Rinder-Serum-Albumin (BSA) auf Siliziumdioxid, Polyelektrolyt-Multischichten (PEM), planaren Polyelektrolyt-Bürsten (PEB) und fluiden sphärischen Polyelektrolyt-Bürsten (SPB) wurden die folgenden Messmethoden in dieser Arbeit angewendet: die Neutronenreflektometrie, die optische Reflektometrie und die CD-Spektroskopie. Adsorption von gelöstem Lysozym und SNase an einer Silizium/Wasser-Grenzfläche Sowohl Lysozym als auch SNase weisen positive Enthalpieänderungen bei ihrer Adsorption aus der Lösung auf oxidiertes Silizium auf. Entropische Kräfte müssen daher bei diesem Vorgang über enthalpische dominieren. Die Wechselwirkung zwischen dem Substrat und den Proteinmolekülen ist attraktiv auf Grund der ungleichnamigen Ladungen. Beide untersuchten Proteine besitzen bei einem pH-Wert von 7 eine positive Nettoladung; das Siliziumdioxid ist demgegenüber negativ geladen. Ein größerer H-Wert bei einer stärkeren Protein-Oberflächenbelegung wird durch eine größere Zahl an repulsiven Protein-Protein-Wechselwirkungen verursacht. Positive Entropieänderungen können zum einen in einer partiellen Dehydratisierung der hydrophoben Substrat- und Proteinoberflächen begründet sein. Zum anderen werden wahrscheinlich durch Protein-Substrat-Wechselwirkungen Lysozym- und SNase-Moleküle partiell entfaltet, so dass deren Konformationsentropie zunehmen sollte. Eine Temperaturerhöhung hat zur Folge, dass die native Proteinstruktur destabilisiert wird und adsorptions-induzierte Konformationsänderungen bevorzugt werden. Proteinadsorption an einer gleichnamig geladenen Polyelektrolyt-Bürste Die variable Proteinbindungskapazität einer planaren PAA-Bürste wurde mit Hilfe der Neutronenreflektometrie und theoretischer Überlegungen charakterisiert. Nachgewiesen werden konnte, dass bei niedriger Ionenstärke große Mengen negativ geladener BSA-Moleküle an einer gleichnamigen PAA-Bürste binden. Bei einer erhöhten Ionenstärke von 0,5 M konnte jedoch mit Hilfe der Neutronenreflektometrie eine Proteinresistenz der Oberfläche beobachtet werden. Diese experimentellen Nachweise entsprechen zuvor durchgeführten Studien an sphärischen PAA-Bürsten-Partikeln. Die Analyse der Neutronenreflektometriekurven zeigt, dass die BSA-Moleküle tief in die PAA-Bürste eindringen. Alle Experimente weisen darauf hin, dass direkte elektrostatische, van der Waals- und hydrophobe Wechselwirkungen nicht für die Proteinaffinität einer PAA-Bürste bei niedriger Ionenstärke verantwortlich sind. Ein einfaches mean field-Modell ist im Rahmen dieser Arbeit entwickelt worden, das den Gewinn von freier Energie durch Freisetzung von Gegenionen, wenn ein Protein-Molekül aus der Lösung in eine gleichnamiggeladene Polyelektrolyt-Bürste eindringt, voraussagt. Diese Gegenionen-„Verdampfung“ ist entropischer Natur und kann über abstoßend wirkende Coulomb-Energien dominieren, wie in dem BSA/PAA-System. Die experimentellen und theoretischen Ergebnisse dieser Studie legen somit eine neue fundamentale Triebkraft für die Proteinadsorption an Grenzflächen nahe. Die Änderung der Ionenstärke einer Proteinlösung im Bereich einiger 100 mM läßt die Struktur und Dynamik eines gelösten Proteinmoleküls unberührt. Das „Schalten“ der Proteinaffinität einer PAA-Bürste durch Änderung der Ionenstärke der Lösung ist wahrscheinlich unabhängig von dem untersuchten Protein, was den Einsatz einer PAA-Bürste in biotechnologischen Anwendung als sehr erfolgsversprechend erscheinen läßt. Zum einen scheint eine einfache und günstige Möglichkeit gegeben, Oberflächen in einem salzhaltigen Medium proteinresistent zu machen. Zum anderen ist die Verwendung als Wirkstoffträger oder Nanoreaktoren in Form von sphärischen Polyelektrolytbürsten denkbar, bei denen die Wirkstoffe (Enzyme) an der Polyelektrolytbürste zunächst immobilisiert und später durch Erhöhung des Salzgehaltes in ihrer Umgebung wieder freigesetzt werden. Besonders wichtig ist hierbei der Aspekt, dass Proteine nach der salzinduzierten Desorption aus der Polyelektrolyt-Bürste ihre Konformation und biologische Aktivität nahezu beibehalten. Adsorption von SNase an einer Polyelektrolyt-Multischicht Polyelektrolyt-Multischichten bieten ein neues Forschungsfeld für die Beschichtung von planaren und kolloiden Substraten. Ihre Herstellung ist einfach. Alternierender Kontakt eines geladenen Substrates mit positiven bzw. negativen Polyelektrolyten führt zu einer selbstanordnenden (self-assembled) Multischicht, die einzigartige Eigenschaften aufweist. Der schrittweise Aufbau eines dreidimensionalen Schichtensystems erlaubt den Einbau einer Vielzahl von Verbindungen und Partikeln, wie Proteinen, Nanopartikeln, Biopolymeren (DNA) und vielen anderen Stoffen. Zu den möglichen Anwendungsbereichen dieser planaren Schichtensysteme zählt der Gebrauch als Matrixmaterial für funktionalisierte oder biologische molekulare Funktionseinheiten, wie Biosensoren, Trennungsmembranen oder ähnliches. In dieser Arbeit wurde die räumliche Verteilung des Proteins SNase, das mit einer planaren Polyelektrolyt-Multischicht interagiert, mit Hilfe neutronenreflektometriescher Messungen bestimmt. Durchgeführt wurden die Messungen bei den Temperaturen 22 °C und 42 °C und bei zwei unterschiedlichen Nettoladungen des Proteins (isoelektrischer Punkt der SNase: 9,5), die durch Wahl des pD-Wertes zu 4,9 bzw. 7,5 erreicht wurde. Die Polyelektrolyt-Multischicht wurde auf einem Silizium-Wafer durch das Auftragen von Polyethylenimin (PEI), Polystyrolsulfonat (PSS) und Polyallylaminhydrochlorid (PAH) in der Reihenfolge Si-PEI-PSS-(PAH-PSS)5 hergestellt. Unter Kontrastvariation wurden für jede Bedingung von Temperatur und pD-Wert zwei unterschiedliche Reflektivitätskurven gemessen. Aus der Analyse der Reflektivitäts-Kurven konnte das Protein-Dichteprofil senkrecht zur Grenzfläche berechnet werden. Die Ergebnisse zeigen, dass die SNase teilweise in die Polyelektrolyt-Multischicht nach der Adsorption bei allen untersuchten Versuchsbedingungen eindringt. Die gemessenen Neutronenreflektivitäten stimmen mit einer Eindringtiefe von 50 Å bei einem pH-Wert von 4,9 und 25 Å bei 7,5 überein. Da SNase einen isoelektrischen Punkt von 9,5 hat, trägt das Protein bei beiden untersuchten pD-Werten eine positive Netto-Ladung und interagiert mit der äußeren letzten PSS-Schicht unter elektrostatisch anziehenden Kräften. Mit ansteigender Temperatur steigt die Menge an adsorbiertem Protein bei beiden pD-Werten an, was die Dominanz einer entropisch treibende Kraft anzeigt. Bei einem pD-Wert von 4,9, bei dem eine relativ hohe Proteinladung vorliegt, ist der temperaturinduzierte Massenanstieg der immobilisierten Proteinmoleküle stärker innerhalb der Polyelektrolytschicht ausgeprägt. Hingegen hat bei einem pD-Wert von 7,5, der näher am isoelektrischen Punkt der SNase liegt, die Erhöhung der Temperatur den Effekt, dass sich die Proteinmoleküle außerhalb der Polyelektrolyt-Multischicht an der Wassergrenzfläche anreichern. Daraus kann geschlossen werden, dass das Eindringen der SNase in eine Polyelektrolyt-Multischicht hauptsächlich auf einem Komplexierungs-mechanismus beruht aufgrund einer Entlassung von Gegenionen. Sphärische Polyelektrolyt-Bürsten als Trägerpartikel für Proteinen Sphärische Polyelektrolyt-Bürsten (SPB) sind eine neue und sehr interessante Art von Kolloid-Partikeln. Sie bestehen aus einem Polystyrolkern, auf dem lange Ketten aus Polyacrylsäure (PAA) oder Polystyrolsäure (PSS) verankert sind. Bei geringer Ionenstärke adsorbieren Proteine spontan auf diesen Partikeln. Eine ihrer besonderen Eigenschaften ist ihre Proteinresistenz bei moderaten Salzkonzentrationen. Daher ist es möglich, durch Erhöhung der Ionenstärke bereits adsorbiertes Protein wieder von den Polyelektrolytketten zu lösen. Dadurch, dass Enzyme auf den SPB geladen und wieder entlassen werden können, bietet sich in Zukunft eine mögliche Anwendung als Wirkstoffträger an. Desweiteren scheint eine mögliche Anwendung als Nanoreaktor, der das immobilisierte Enzym nach einer gewünschten Reaktion wieder aus den SPB entlassen kann, sehr erfolgsversprechend. Mit Hilfe der CD-Spektroskopie wurde in dieser Arbeit die Konformation eines Proteins, welches mit einer sphärischen Polyelektrolytbürste wechselwirkt, untersucht. Rinder-Serum-Albumin (BSA) wurde als Modellprotein gewählt. Es trägt bei neutralen pH-Werten aufgrund seines isoelektrischen Punktes von pH=5 eine negative Nettoladung und besteht in seiner Sekundärstruktur größtenteils aus α-Helices. Die Ergebnisse der CD-Messungen zeigen, dass BSA durch Wechselwirkung mit SPB, d.h. nach einem Adsorptions-Desorptions-Prozess, nur geringfügig seine Sekundärstruktur verändert. Dabei stehen die beobachteten Konformationsänderungen nicht in Zusammenhang mit denen, die in Lösung bei niedrigen pH-Werten gefunden werden. SPB erweisen sich somit als vielversprechende Trägerpartikel für Proteine, da sie eine schonende Proteinumgebung bereit stellen.

In this thesis we study the kinetics of fibrilar aggregation of two model proteins widely used in the food industry -b-lactoglobulin (b-lg) and hen egg white lysozyme (HEWL). The kinetics of protein aggregation is studied mostly experimentally and, when possible, theoretically. The process of fibrilar (or linear) protein aggregation is the process of formation of elongated structures from otherwise compact (globular) proteins. Studying the kinetics of this process for different proteins can lead to a better understanding of the mechanism of the process and to a possible generalisation of this mechanism. The investigation of the morphology of the formed aggregates at different stages of the process of aggregation could also lead to a more complete picture of the detailed mechanism of the process. Last, but not least, is the influence of the protein stability on the type of the formed aggregates and the kinetics of fibrilar aggregation.The specific aims of this thesis are the following: 1) To investigate the kinetics of heat-induced fibrilar aggregation of two model proteins, bovineb-lg and HEWL, in as much detail as possible; 2) To study the morphology of the fibrils formed from both proteins; 3) To study the influence of the environment such as temperature, pH, and ionic strength on the kinetics of fibrilar aggregation and the morphology of the formed fibrils.The heat-induced fibrilar aggregation ofb-lg is investigated at pH 2.0, 80°C, and at various ionic strengths. Fibril formation is followed in situ using static (SLS) and dynamic light scattering (DLS), small angle neutron scattering (SANS), and proton NMR techniques. The fibrils that form after short heating periods (up to a few hours) disintegrate upon slow cooling, whereas fibrils that form during long heating periods do not disintegrate upon subsequent slow cooling. Even after prolonged heating, an appreciable fraction of the protein molecules is incorporated into fibrils, only when theb-lg concentration is above some critical concentration that is ionic strength dependent.The linear aggregation ofb-lg upon prolonged heating at pH 2.0 at80°Cappears to be a multistep process. Competing reactions lead to two products: long linear aggregates and low molecular weight "dead end" species. The "dead end" species comprises monomeric non-native protein molecules and cannot form fibrils. Fibril formation involves at least two steps: the reversible formation of linear aggregates, followed by a slow process of "consolidation" after which the fibrils no longer disintegrate upon subsequent slow cooling.Based on the obtained experimental data we have derived a kinetic model for the heat-induced aggregation ofb-lg at pH 2.0. The model involves a nucleation step and a simple addition reaction for the growth of the fibrils as well as a side reaction leading to the complete denaturation and inactivation of a part of the protein molecules. An analytical solution of the model for the early stages of the aggregation is obtained. The model describes very well the experimental data obtained by in situ SLS. It allows us to obtain molecular parameters for the kinetics of fibrilar aggregation ofb-lg as a function of the ionic strength. It gives us an expression for the apparent critical concentration for fibril formation due to the competition between the complete denaturation of the protein molecules and the formation of long fibrils. We also obtain the size of the critical nucleus for the fibril formation as a function of the ionic strength. In the case of a 13 mM ionic strength the critical nucleus consists of ca. 4 monomers; for all the other ionic strengths studied it is a dimer. This shows the important role that the non-specific electrostatic interaction has for the fibrilar aggregation ofb-lg at pH 2.0. It affects the rate of aggregation: the higher the ionic strength, the faster the aggregation. It also affects the detailed mechanism by which the aggregation takes place: the size of the critical nucleus increases when decreasing the ionic strength from 50 mM to 13 mM.We have also shown that time-resolved SANS can be used with success in studying protein aggregation and that with enough additional information for the aggregation process one can in practice obtain complete information about the aggregation kinetics of the process.Tapping mode atomic force microscopy results indicate that the fibrils formed at pH 2.0 upon heating at 80°Chave a periodic structure with a period of about 25 nm and a thickness of one or two protein monomers. The main difference between the fibrils observed at different ionic strengths is their length and curvature. Fibrils obtained at higher ionic strength are shorter and more curved as opposed to longer and straighter fibrils obtained at lower ionic strengths. In case of higher ionic strength the fibril formation is faster, more fibrils are formed and as a result the mean length of the fibrils is shorter. Fibrils obtained at all ionic strengths exhibit similar type of periodic morphology, which suggests that the detailed mechanism of fibril formation might be independent of the ionic strength, but specific forb-lg.In the case of HEWL we study the effect of pH and temperature on the fibril formation. Fibril formation is promoted by low pH and temperatures close to the midpoint temperature for protein unfolding (detected using far-ultraviolet circular dichroism (CD)). The stability of HEWL toward heat treatment is greatly influenced by the pH. The lower the pH, the lower the stability of the protein is. The conditions at pH 2.0 are unique in promoting the fibrilar aggregation of HEWL since heating of solutions at pH 3.0 and 4.0 to temperatures just above the midpoint of the unfolding transition of the molecule does not lead to the appearance of fibrilar aggregates.HEWL fibrils are formed after a lag time that is practically concentration independent. This means that the governing process for the fibril formation is the change in the structure of single protein molecules caused by a prolonged exposure to a temperature close to the midpoint of the unfolding transition. Nucleation presumably involves a change in the conformation of individual lysozyme molecules. Indeed, long term CD measurements at pH 2.0, T = 57°C show a marked change of the secondary structure of lysozyme molecules after about 48 h of heating.The fibril morphology is complex. The fibrils formed at pH 2.0 are long and straight with a length of the order of 5mm and predominant thickness of about 4 nm and consist of stiff rod-like subunits with length either 124 or 157 nm. On smaller scale the fibrils consist of a coiled structure with a period of ca. 30 nm that gives the appearance of the rod-like subunits probably because of defects occurring every 4 or 5 turns.The fibrils consist mostly of full-length HEWL, although, some fragments due to hydrolysis at pH 2.0 and 57°C are probably incorporated into the fibrils. At any rate the hydrolysis of the protein is not the cause of the aggregation since at pH 3.0 no hydrolysis is detected but fibrils do form.In conclusion we can say that for a full and general description of the processes of fibrilar aggregation of globular proteins the type of specific interaction responsible for the aggregation must be identified. The interacting parts of the protein must also be identified. The last and most difficult task is to characterise the conformation of the protein in solution at conditions suitable for aggregation.

In this thesis we study the kinetics of fibrilar aggregation of two model proteins widely used in the food industry -b-lactoglobulin (b-lg) and hen egg white lysozyme (HEWL). The kinetics of protein aggregation is studied mostly experimentally and, when possible, theoretically. The process of fibrilar (or linear) protein aggregation is the process of formation of elongated structures from otherwise compact (globular) proteins. Studying the kinetics of this process for different proteins can lead to a better understanding of the mechanism of the process and to a possible generalisation of this mechanism. The investigation of the morphology of the formed aggregates at different stages of the process of aggregation could also lead to a more complete picture of the detailed mechanism of the process. Last, but not least, is the influence of the protein stability on the type of the formed aggregates and the kinetics of fibrilar aggregation.The specific aims of this thesis are the following: 1) To investigate the kinetics of heat-induced fibrilar aggregation of two model proteins, bovineb-lg and HEWL, in as much detail as possible; 2) To study the morphology of the fibrils formed from both proteins; 3) To study the influence of the environment such as temperature, pH, and ionic strength on the kinetics of fibrilar aggregation and the morphology of the formed fibrils.The heat-induced fibrilar aggregation ofb-lg is investigated at pH 2.0, 80°C, and at various ionic strengths. Fibril formation is followed in situ using static (SLS) and dynamic light scattering (DLS), small angle neutron scattering (SANS), and proton NMR techniques. The fibrils that form after short heating periods (up to a few hours) disintegrate upon slow cooling, whereas fibrils that form during long heating periods do not disintegrate upon subsequent slow cooling. Even after prolonged heating, an appreciable fraction of the protein molecules is incorporated into fibrils, only when theb-lg concentration is above some critical concentration that is ionic strength dependent.The linear aggregation ofb-lg upon prolonged heating at pH 2.0 at80°Cappears to be a multistep process. Competing reactions lead to two products: long linear aggregates and low molecular weight "dead end" species. The "dead end" species comprises monomeric non-native protein molecules and cannot form fibrils. Fibril formation involves at least two steps: the reversible formation of linear aggregates, followed by a slow process of "consolidation" after which the fibrils no longer disintegrate upon subsequent slow cooling.Based on the obtained experimental data we have derived a kinetic model for the heat-induced aggregation ofb-lg at pH 2.0. The model involves a nucleation step and a simple addition reaction for the growth of the fibrils as well as a side reaction leading to the complete denaturation and inactivation of a part of the protein molecules. An analytical solution of the model for the early stages of the aggregation is obtained. The model describes very well the experimental data obtained by in situ SLS. It allows us to obtain molecular parameters for the kinetics of fibrilar aggregation ofb-lg as a function of the ionic strength. It gives us an expression for the apparent critical concentration for fibril formation due to the competition between the complete denaturation of the protein molecules and the formation of long fibrils. We also obtain the size of the critical nucleus for the fibril formation as a function of the ionic strength. In the case of a 13 mM ionic strength the critical nucleus consists of ca. 4 monomers; for all the other ionic strengths studied it is a dimer. This shows the important role that the non-specific electrostatic interaction has for the fibrilar aggregation ofb-lg at pH 2.0. It affects the rate of aggregation: the higher the ionic strength, the faster the aggregation. It also affects the detailed mechanism by which the aggregation takes place: the size of the critical nucleus increases when decreasing the ionic strength from 50 mM to 13 mM.We have also shown that time-resolved SANS can be used with success in studying protein aggregation and that with enough additional information for the aggregation process one can in practice obtain complete information about the aggregation kinetics of the process.Tapping mode atomic force microscopy results indicate that the fibrils formed at pH 2.0 upon heating at 80°Chave a periodic structure with a period of about 25 nm and a thickness of one or two protein monomers. The main difference between the fibrils observed at different ionic strengths is their length and curvature. Fibrils obtained at higher ionic strength are shorter and more curved as opposed to longer and straighter fibrils obtained at lower ionic strengths. In case of higher ionic strength the fibril formation is faster, more fibrils are formed and as a result the mean length of the fibrils is shorter. Fibrils obtained at all ionic strengths exhibit similar type of periodic morphology, which suggests that the detailed mechanism of fibril formation might be independent of the ionic strength, but specific forb-lg.In the case of HEWL we study the effect of pH and temperature on the fibril formation. Fibril formation is promoted by low pH and temperatures close to the midpoint temperature for protein unfolding (detected using far-ultraviolet circular dichroism (CD)). The stability of HEWL toward heat treatment is greatly influenced by the pH. The lower the pH, the lower the stability of the protein is. The conditions at pH 2.0 are unique in promoting the fibrilar aggregation of HEWL since heating of solutions at pH 3.0 and 4.0 to temperatures just above the midpoint of the unfolding transition of the molecule does not lead to the appearance of fibrilar aggregates.HEWL fibrils are formed after a lag time that is practically concentration independent. This means that the governing process for the fibril formation is the change in the structure of single protein molecules caused by a prolonged exposure to a temperature close to the midpoint of the unfolding transition. Nucleation presumably involves a change in the conformation of individual lysozyme molecules. Indeed, long term CD measurements at pH 2.0, T = 57°C show a marked change of the secondary structure of lysozyme molecules after about 48 h of heating.The fibril morphology is complex. The fibrils formed at pH 2.0 are long and straight with a length of the order of 5mm and predominant thickness of about 4 nm and consist of stiff rod-like subunits with length either 124 or 157 nm. On smaller scale the fibrils consist of a coiled structure with a period of ca. 30 nm that gives the appearance of the rod-like subunits probably because of defects occurring every 4 or 5 turns.The fibrils consist mostly of full-length HEWL, although, some fragments due to hydrolysis at pH 2.0 and 57°C are probably incorporated into the fibrils. At any rate the hydrolysis of the protein is not the cause of the aggregation since at pH 3.0 no hydrolysis is detected but fibrils do form.In conclusion we can say that for a full and general description of the processes of fibrilar aggregation of globular proteins the type of specific interaction responsible for the aggregation must be identified. The interacting parts of the protein must also be identified. The last and most difficult task is to characterise the conformation of the protein in solution at conditions suitable for aggregation.