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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers
Isoelectric points of proteins at the air/liquid interface and in solution Tobias Guckeisen, Saman Hosseinpour, and Wolfgang Peukert Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00311 • Publication Date (Web): 20 Mar 2019 Downloaded from http://pubs.acs.org on March 22, 2019
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Isoelectric points of proteins at the air/liquid interface and in solution Tobias Guckeisen, Saman Hosseinpour, Wolfgang Peukert Institute of Particle Technology (LFG), Friedrich-Alexander-Universität-Erlangen-Nürnberg (FAU), Cauerstraße 4, 91058 Erlangen, Germany Proteins, Isoelectric point (IEP), Isoelectric point at the surface (IEPS), Sum frequency generation spectroscopy, Zeta potential, Ellipsometry, air/water interface
ABSTRACT: Electrostatic interactions play essential roles in determining the function, colloidal stability, and adsorption of proteins on different surfaces and interfaces. Therefore, a molecular level understanding of the charge state of the proteins under different conditions is required to explain their macroscopic properties. In this study we have employed an inherently surface sensitive spectroscopic tool, sum frequency generation spectroscopy, to determine the charge state of a wide range of proteins as a function of pH at the liquid/air interface via measurement of the degree of orientation of water molecules. We compared the isoelectric point of the 12 investigated proteins at the liquid/air interface with that in the bulk solution obtained through zeta potential measurements. Ellipsometry is performed to determine the film thickness at the liquid/air interface at different charge states. In particular, protein aggregation at the isoelectric
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point is reflected by increased film thickness. For all proteins, the interfacial point of zero charge is close (with less than 1 pH unit variation) to that in the bulk solution.
Introduction Electrostatic interactions are amongst the most important factors that determine the functionality, thermal and colloidal stability, complex formation, solubility, and adsorption behavior of proteins. The electrostatic properties of proteins are regulated by the distribution and ratio of charged and polar residues within the proteins structure. Environmental factors such as temperature, hydrophobicity, and pH of the surrounding medium may alter the distribution of the charged and polar residues in proteins and thus affect their electrostatic properties. Understanding the charge state of proteins in a molecular level under different conditions is thus vital in explaining their macroscopic properties. Proteins exhibit an overall positive, negative or neutral charge state depending on their surrounding environment as the ionization state of the proteins’ main amino acid groups vary with environmental factors such as solution pH. Accordingly, repulsive or attractive electrostatic forces act between the similarly or oppositely charged proteins. When the overall protein charge approaches zero, the acting electrostatic forces diminish, the solubility of protein decreases, and the other forces such as hydrogen bonding, hydrophobic interactions, and van der Waals and steric interactions become dominant in determining the stability of the proteins. The pH in which the overall charge state of a protein is minimum is denoted as isoelectric point (IEP). In fact, many of the protein separation and purification techniques such as electrophoresis or ion exchange chromatography rely on the IEP of proteins.
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Therefore, many experimental and theoretical methods have been developed to determine the isoelectric point of proteins.1 The IEP of proteins in the bulk solution is measured routinely through isoelectric focusing2,3 or by measuring the electrophoretic mobility.4 There is also a possibility to estimate the isoelectric point from the amino acid sequence by a range of methods readily available for such calculations.5,6 However, the calculated IEP values using different approaches vary up to several pH units.5 Moreover, it should be noted that the calculated isoelectric points are in fact relevant to the proteins charge state in pure water and should be referred to as the isoionic point. The isoionic point is defined as the pH value at which a zwitterionic molecule has an equal number of positive and negative charges and no adsorbed ion species.7,8 It has been shown that the measured isoelectric point is affected by the ion concentration, ion species, or buffer conditions,8,9 which limits the credibility of the calculated isoionic points of proteins in the biologically relevant conditions. Proteins tend to aggregate especially at the interfaces in combination with interfacial stresses.10,11 Lately, the importance of protein-protein interactions to predict protein aggregation at the interface has been emphasized.12 The protein interactions near the IEP are most favorable for aggregation at the interface or in the bulk due to the lack of a net charge and therefore a lack of electrostatic stabilization. Thus, the isoelectric point at the surface (IEPS) is of outstanding relevance for interfacial aggregation processes. IEPS plays a crucial role for protein foaming and foam stabilities,13,14 where aggregates can have a stabilizing effect, and where the molecular structure of the interface controls macroscopic foam properties including foam stability and rheology. Another example is in protein drugs such as insulin15 and antibodies,11 where aggregates are highly undesirable and interfaces are known as contributing factors in aggregation process.11 Additionally, since proteins are technically produced in aerated bioreactors such as
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bubble columns, the behavior of proteins at the bubbles (solution/air interface) is very important as well. Proteins in tear fluid16 or lung fluids are further examples where layered structures may form at the liquid/air interface. Recently, sum frequency generation spectroscopy (SFG) has been employed to determine the IEP of selected model proteins at the liquid/air interface.17 Theoretical and experimental aspects of SFG have been provided elsewhere,18–21 and here only the necessary details are explained. Briefly, in SFG spectroscopy a visible laser beam with a fixed wavelength (ωvis) overlaps spatially and temporally with a tunable laser beam in the infrared frequency region (ωIR). At the surfaces and interfaces where the bulk symmetry is broken, a SF radiation is generated with its frequency being the sum of the frequencies of the incoming beams (ωSFG=ωvis+ωIR). Under the dipole approximation, in centrosymmetric bulk media with randomly distributed dipoles no SFG signal is generated, granting SFG inherent surface sensitivity. The intensity of the SFG signal is a function of number density of the aligned molecules at the interface. At the electrified surfaces polar water molecules are preferentially ordered with O or OH aligned toward the positively or negatively charged surfaces, respectively, enhancing the SFG signal in the OH stretching frequency region (3100 cm-1 - 3750cm-1). Therefore, the SFG signal intensity in this frequency region can be used as a quantitative measure of the amount of charge at the electrified surfaces. In fact, this principle is used in determining the IEP of the proteins at the liquid/air interface: the SFG signal intensity in the OH stretching region approaches its minimum close to the IEPS of proteins. The conformation of proteins and their electrostatic properties at the solution/air interface might be very different from those in the bulk solution. Since the actual charge and pKs values of amino acids are dependent on the environment and as proteins at the interface reside in a very
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different environment than in the bulk, it may be expected that the interfacial IEP is different from the bulk IEP. Therefore, knowledge of the IEP of proteins at the liquid/air interface becomes very crucial in understanding the interfacial behaviors of proteins. In this study, we utilize SFG to measure the IEPS of a wide range of proteins with different sizes, hydrophobicities, and structural elements. We then compare the measured interfacial IEPs with those measured in the bulk through zeta potential measurements. To correlate the IEPs to the macroscopic properties of the studied proteins, ellipsometry is performed under similar conditions as the IEPS measurements. This approach enables us to provide a unified picture of how charge states at the proteins in the bulk solution and at the liquid/air interface will be translated to their macroscopic properties, e.g. film thickness in this case. Materials and Methods Sample Preparation. Bovine Serum Albumin (BSA; A7030), Ribonuclease A (RNase A; R5503), Chymotrypsinogen A (Chym A; C4879), Catalase (SRE 0041), Ovalbumin (A5503), Cytochrome C (Cyt C; C2506), α-lactalbumin (LALBA; L6010), Hemoglobin (Hem; H7379) and Insulin (I2643) were purchased from Sigma-Aldrich. Lysozyme (Lys; 8259.2) was purchased from Carl Roth and anti-freezeprotein type III (AFP III) was purchased from A/F Protein inc. All proteins was used as received with no further purification. β-lactoglobulin (BLG) which was isolated as described previously22 was kindly provided by the group of Ulrich Kulozik (Technische Universität München, Germany). The protein powders were dissolved in Milli-Q water (18.2 MΩcm, total oxidizable carbon
