Infrared absorbance spectroscopy of aqueous proteins: Comparison of transmission and ATR data collection and analysis for secondary structure fitting

Attenuated total reflectance (ATR) infrared absorbance spectroscopy of proteins in aqueous solution is much easier to perform than transmission spectroscopy, where short path‐length cells need to be assembled reproducibly. However, the shape of the resulting ATR infrared spectrum varies with the refractive index of the sample and the instrument configuration. Refractive index in turn depends on the absorbance of the sample. In this work, it is shown that a room temperature triglycine sulfate detector and a ZnSe ATR unit can be used to collect reproducible spectra of proteins. A simple method for transforming the protein ATR spectrum into the shape of the transmission spectrum is also given, which proceeds by approximating a Kramers‐Krönig–determined refractive index of water as a sum of four linear components across the amide I and II regions. The light intensity at the crystal surface (with 45° incidence) and its rate of decay away from the surface is determined as a function of the wave number–dependent refractive index as well as the decay of the evanescent wave from the surface. The result is a single correction factor at each wave number. The spectra were normalized to a maximum of 1 between 1600 cm^?1 and 1700 cm^?1 and a self‐organizing map secondary structure fitting algorithm, SOMSpec, applied using the BioTools reference set. The resulting secondary structure estimates are encouraging for the future of ATR spectroscopy for biopharmaceutical characterization and quality control applications.     

Application of Fourier transform infrared spectroscopy to studies of aqueous protein solutions.

Modern protein Fourier transform infrared (FT-IR) spectroscopy has proven to be a versatile and sensitive technique, applicable to many aspects of protein characterization. The major practical drawback for the FT-IR spectroscopy of proteins is the large absorbance band of water, which overlaps the amide I resonances. D2O is often substituted for H2O in infrared experiments. Removal of water from protein samples can be complicated and tedious and potentially lead to denaturation, aggregation, or sample loss. Solvent removal by dialysis is difficult for suspensions and sols. A new method called the D2O dilution technique (Ddt) is described which simplifies the sample preparation step and improves the solvent subtraction. The effect of the D2O concentration on the IR spectrum of aqueous solutions of several model proteins was studied. Dilution of aqueous samples with D2O yields good quality spectra. The Ddt has been evaluated for quantitative analysis using standard proteins and its applicability to solutions and suspensions of a genetically engineered malaria antigen is demonstrated. Use of resolution-enhancement techniques with spectra in mixed solvents has also been investigated.     

Rapid measurement of phytate in raw soymilk by mid-infrared spectroscopy

The phytate content in soymilk is known to affect tofu curdling. A rapid measurement of phytate from a water extract of soybean (raw soymilk) in an early stage of tofu processing was investigated using mid-infrared spectroscopy (IR) with an ATR accessory. IR absorption of phytate was observed from 1200 cm-1 to 900 cm-1, and saccharide and protein in the extract also had IR absorption in the same region. In order to separate phytate from other components, the phytate was precipitated completely by the addition of calcium under alkaline condition (pH 11.5). The precipitate was dissolved in citrate buffer (pH 6.0) and then used for IR measurement. The absorbance at 1070 cm-1 correlated well with the phytate content of the soymilk. The measurement of phytate in raw soymilk can be done rapidly by FT-IR measurement with an ATR accessory and gives reproducible values, which can be used for the measurement of phytate content in various soybeans for tofu making.     

Conformational changes in bacteriorhodopsin studied by infrared attenuated total reflection.

We report on a new method based on Fourier transform infrared (FTIR)-difference spectroscopy for studying the conformational changes occurring during the photocycle of bacteriorhodopsin. Previous studies have been made by measuring the absorbance of an infrared (IR) beam transmitted through a thin hydrated purple membrane film. In contrast, the present study utilizes the technique of attenuated total reflection (ATR). Purple membrane is fixed on the surface of a germanium internal reflection crystal and immersed in a buffer whose pH and ionic composition can be varied. Measurements of the amide I and II absorbance with light polarized parallel and at 45 degrees to the crystal surface reveals that the membrane is highly oriented. An ATR-FTIR-difference spectrum of the light to dark (bR570 to bR548) transition is similar but not identical to the transmittance FTIR-difference spectrum. This disagreement between the two methods is shown to be due in the ATR case to the absorption of transition moments oriented predominantly out of the membrane plane. Raising the pH of La3+ substituted purple membrane films from 6.8 to 8.0 slows the M-decay rate sufficiently so that a bR570 to M412 difference spectrum can be obtained with steady state illumination at room temperature. A comparison of this difference spectrum with that obtained at -23 degrees C using the transmittance method reveals several changes that cannot be attributed to out-of-plane transition moments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative surface studies of protein adsorption by infrared spectroscopy. I. Correction for bulk concentrations

A method for correcting attenuated total reflectance (ATR) infrared spectra of surface-adsorbed proteins for the contribution of proteins in the bulk solution has been demonstrated. This procedure estimates the soluble protein contribution to the ir spectra from parameters determined from transmission experiments, and uses the absorbance of the water band in ATR as an internal reference. Based on this procedure, the soluble protein contribution to the total spectrum is estimated to range from approximately 3% for IgG or albumin in solution at 1 mg/ml, to approximately 35% when whole blood (from sheep) is adsorbed onto a 45 degrees germanium ATR crystal.     

Surface analysis of sheep menisci after meniscectomy via infrared attenuated total reflection spectroscopy

Menisci are very important fibrocartilaginous tissue, which maintain biomechanical functions and physiological stabilization of knee joint. Meniscectomy is known as a surgery to recover partial functions from acute meniscus tears. However, the late consequences of total or partial meniscectomy include signs of osteoarthritis and even ligament instability. Infrared attenuated total reflection (IR‐ATR) spectroscopy is a very useful technique, which can reveal molecular characteristics via the analysis of vibrational bands. The present study has employed IR‐ATR spectroscopy to investigate sheep menisci samples after meniscectomy in a label‐free fashion. Several differences of peak absorbance change and peak shift were observed between the native healthy samples and the meniscectomy samples in distinct IR wavenumber regions, such as amide I band, amide II band, C‐H bending band as well as the sugar band region. Combining the results from the collagen protein IR spectra, it can be speculated that six months after meniscectomy collagen fibrils on the incision lose its ordered arrangement and a decrease in the triple helical structure of collagen fibril is observed. In addition, the collagen fibrils and proteoglycan content might also be slight varied after meniscectomy.      

In situ FTIR ATR spectroscopic study of the interaction of immobilized human tumor necrosis factor-alpha with a monoclonal antibody in aqueous environment

By in situ FTIR ATR measurements, the antibody (AB) recognition of human tumor necrosis factor-alpha (TNFalpha) immobilized on the Ge surface of a multiple internal reflection element (MIRE) was investigated. The experiments were performed in aqueous environment in a flow-through cell. After immobilization of TNFalpha on the Ge-MIRE by direct adsorption from aqueous solution, the immobilisate reached stability after about 1 h under flow-through conditions. The remaining sites of the Ge surface were saturated by bovine serum albumin (BSA) in order to prevent unspecific binding of anti-TNFalpha AB which was then added. The obtained FTIR ATR spectra were shown to result exclusively from AB specifically interacting with TNFalpha, since the absence of immunoglobulin binding to BSA adsorbed to the Ge MIRE was verified by a reference experiment. Finally, the stability of all adsorbed protein immobilisates was monitored under flow-through conditions for 10.5 h. The TNFalpha-AB complex showed a decrease of 7.4%, whereas the BSA adsorbate remained stable. IR measurements were performed with polarized light in order to study orientational effects of the immobilized proteins. The dichroic ratios and surface concentrations of all used proteins are available after quantitative analysis of the amide II bands.     

Infrared study of trifluoroacetic acid unpurified synthetic peptides in aqueous solution: trifluoroacetic acid removal and band assignment

Synthetic peptide or protein samples are mostly unpurified with trifluoroacetic acid (TFA) used during the synthesis procedure, which strongly interferes with structure determination by infrared (IR) spectroscopy. The aim of this work was to propose a simple strategy to remove TFA contribution from attenuated total reflection (ATR)–IR spectra of the hexahistidine peptide (His6) in aqueous solution to study the conformation of this synthetic peptide without previous purification. Such a strategy is based on the subtraction mode widely employed to remove water contribution, and it is tested with TFA unpurified histidine as a model system. The subtraction is based on eliminating the strong TFA bands at 1147 and 1200 cm⁻¹ by applying a scaling factor (as in buffer correction). The proposed modes represent excellent strategies that do not modify spectral features, and they provide reliable routines to obtain the synthetic peptide spectrum without TFA contribution. The conformational information from the corrected spectra at different pH values is deduced from semiempirical calculated IR spectra of different His6 conformers. The spectral features and the band positions of the corrected spectrum suggest that the peptide molecules mainly adopt an intermolecular β-sheet structure.     

Rapid activation of ATR by ionizing radiation requires ATM and Mre11

The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases are crucial regulatory proteins in genotoxic stress response pathways that pause the cell cycle to permit DNA repair. Here we show that Chk1 phosphorylation in response to hydroxyurea and ultraviolet radiation is ATR-dependent and ATM- and Mre11-independent. In contrast, Chk1 phosphorylation in response to ionizing radiation (IR) is dependent on ATR, ATM, and Mre11. The ATR and ATM/Mre11 pathways are generally thought to be separate with ATM activation occurring early and ATR activation occurring as a late response to double strand breaks. However, we demonstrate that ATR is activated rapidly by IR, and ATM and Mre11 enhance ATR signaling. ATR-ATR-interacting protein recruitment to double strand breaks is less efficient in the absence of ATM and Mre11. Furthermore, IR-induced replication protein A foci formation is defective in ATM- and Mre11-deficient cells. Thus, ATM and Mre11 may stimulate the ATR signaling pathway by converting DNA damage generated by IR into structures that recruit and activate ATR.     

Recent applications of ATR FTIR spectroscopy and imaging to proteins

Attenuated Total Reflection (ATR) Fourier Transform Infrared (FTIR) spectroscopy is a label-free, non-destructive analytical technique that can be used extensively to study a wide variety of different molecules in a range of different conditions. The aim of this review is to discuss and highlight the recent advances in the applications of ATR FTIR spectroscopic imaging to proteins. It briefly covers the basic principles of ATR FTIR spectroscopy and ATR FTIR spectroscopic imaging as well as their advantages to the study of proteins compared to other techniques and other forms of FTIR spectroscopy. It will then go on to examine the advances that have been made within the field over the last several years, particularly the use of ATR FTIR spectroscopy for the understanding and development of protein interaction with surfaces. Additionally, the growing potential of Surface Enhanced Infrared Spectroscopy (SEIRAS) within this area of applications will be discussed. The review includes the applications of ATR FTIR imaging to protein crystallisation and for high-throughput studies, highlighting the future potential of the technology within the field of protein structural studies and beyond.     

Fourier transform infrared studies of proteins using nonaqueous solvents. Effects of methanol and ethylene glycol on albumin and immunoglobulin G

An infrared/attenuated total reflection (ATR) technique has been utilized to study the structural changes in proteins induced by nonaqueous solvents, without the need of dissolving the protein in the nonaqueous solvent. For the two proteins studied, methanol and ethylene glycol caused similar changes in albumin, i.e., an increase in helix secondary structure. However, the two solvents had dissimilar effects on immunoglobulin G (IgG). Changes in the pH of aqueous solutions of IgG produced a third effect. By dissolving some IgG in ethylene glycol and then adsorbing IgG from this solution onto an ATR crystal, the time behavior of the adsorption process could be studied and a mechanism for the structural changes proposed.     

Easily polarizable N+H…N hydrogen bonds between histidine side chains and proton translocation in proteins

Histidinium perchlorate having protecting groups at the α-amino and α-carboxylate group is studied by IR spectroscopy as function of the addition of protected histidine molecules. An intense continuous absorption arises, indicating that the N⁺H…N ? N…H⁺N formed are easily polarizable hydrogen bonds. From the integral absorbance of a band the concentration of the histidine-histidinium complex, i.e. the concentration of the easily polarizable hydrogen bonds is determined. It is shown that the absorbance of the continuum increases in proportion to the concentration of the easily polarizable N⁺H…N ? N…H⁺N bonds. Finally, it is discussed that via such an easily polarizable histidine-histidinium hydrogen bond a proton translocation in the active center of ribonuclease A may occur.     

Protein structural perturbation and aggregation on homogeneous surfaces

We have demonstrated that globular proteins, such as hen egg lysozyme in phosphate buffered saline at room temperature, lose native structural stability and activity when adsorbed onto well-defined homogeneous solid surfaces. This structural loss is evident by alpha-helix to turns/random during the first 30 min and followed by a slow alpha-helix to beta-sheet transition. Increase in intramolecular and intermolecular beta-sheet content suggests conformational rearrangement and aggregation between different protein molecules, respectively. Amide I band attenuated total reflection/Fourier transformed infrared (ATR/FTIR) spectroscopy was used to quantify the secondary structure content of lysozyme adsorbed on six different self-assembled alkanethiol monolayer surfaces with -CH3, -OPh, -CF3, -CN, -OCH3, and -OH exposed functional end groups. Activity measurements of adsorbed lysozyme were in good agreement with the structural perturbations. Both surface chemistry (type of functional groups, wettability) and adsorbate concentration (i.e., lateral interactions) are responsible for the observed structural changes during adsorption. A kinetic model is proposed to describe secondary structural changes that occur in two dynamic phases. The results presented in this article demonstrate the utility of the ATR/FTIR spectroscopic technique for in situ characterization of protein secondary structures during adsorption on flat surfaces.     

A quantitative and selective chromatography method for determining coverages of multiple proteins on surfaces

Competitive protein adsorption plays a key role in the surface hemocompatibility of biological implants. We describe a quantitative chromatography method to measure the coverage of multiple proteins physisorbed to surfaces. In this method adsorbed proteins are displaced by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and then analyzed by high performance liquid chromatography to separate and quantify the individual proteins, in this case bovine serum albumin (BSA) and bovine fibrinogen (Fg). CHAPS displaced over 95% of the adsorbed proteins and was easily removed from solution by dialysis. This method was tested by measuring the coverage of BSA, 66 kDa, and Fg, 340 kDa, simultaneously adsorbed from solutions with concentration of 20 microg/ml, on bare and dextranized silicon. Relative to silicon, the dextranized surfaces were found to strongly inhibit protein adsorption, decreasing BSA and Fg coverages by 76 and 60%, respectively.     

Rapid analysis of disease state in liquid human serum combining infrared spectroscopy and “digital drying”

In recent years, the diagnosis of brain tumors has been investigated with attenuated total reflection‐Fourier transform infrared (ATR‐FTIR) spectroscopy on dried human serum samples to eliminate spectral interferences of the water component, with promising results. This research evaluates ATR‐FTIR on both liquid and air‐dried samples to investigate “digital drying” as an alternative approach for the analysis of spectra obtained from liquid samples. Digital drying approaches, consisting of water subtraction and least‐squares method, have demonstrated a greater random forest (RF) classification performance than the air‐dried spectra approach when discriminating cancer vs control samples, reaching sensitivity values higher than 93.0% and specificity values higher than 83.0%. Moreover, quantum cascade laser infrared (QCL‐IR) based spectroscopic imaging is utilized on liquid samples to assess the implications of a deep‐penetration light source on disease classification. The RF classification of QCL‐IR data has provided sensitivity and specificity amounting to 85.1% and 75.3% respectively.     

Simultaneous multiple element detection by particle beam/hollow cathode-optical emission spectroscopy as a tool for metallomic studies: determinations of metal binding with apo-transferrin

Particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) is presented as a tool for the determination of metal ion loading in transferrin (Tf). The elemental specificity of optical emission spectroscopy provides a means of assessing metal ion concentrations as well as the relative amounts of metal per unit protein concentration (up to 2 moles of Fe per mole of protein). The PB/HC-OES method allows for the simultaneous detection of metal content (Fe (I) 371.99, Ni (I) 341.41 nm, Zn (I) 213.86 nm, and Ag (I) 338.28 nm in this case), as well as elemental carbon and sulfur (C (I) 156.14 nm and S (I) 180.73 nm) that are reflective of the protein composition and concentration. Quantification for the metal species is based on calibration functions derived from aqueous solutions, with limits of detection for the entire suite being less than 1.0 μM. Determinations in this manner eliminate much of the ambiguity inherent in UV-VIS absorbance determinations of Tf metal binding. Validation of this method is obtained by analyzing loading response of Fe(3+) into Tf using the PB/HC-OES method and comparing the results with those of the standard UV-VIS absorbance method. Maximum Fe(3+) loading of Tf (based on the number of available binding sites) was determined to be 71.2 ± 4.7% by the PB/HC-OES method and 67.5 ± 2.5% for the UV-VIS absorbance method. Element emission ratios between the dopant metals and the carbon and sulfur protein constituents allow for concentration independent determinations of metal binding into Tf. Loading percentages were determined for Ni(2+), Zn(2+), and Ag(+) into Tf with maximum loading values of 19.5 ± 0.4%, 41.0 ± 4.4%, and 141.2 ± 4.3%, respectively. While of no apparent biological significance, Ag(+) presents an interesting case as a surrogate for Pt(2+), whose binding with Tf has shown to be quite different from the other metals. A different mode from the others is indeed observed, and is consistent with conjecture on the Pt(2+) mechanisms. Competitive binding studies not easily performed using absorbance spectroscopy are easily performed by simultaneous, multielement analysis, reflective of the metals and the protein content. In this work, there is clear competition between and Fe(3+) and Zn(2+) for binding in the C-terminus lobe of Tf, while Ni(2+) binds within the N-terminus lobe. Addition of Ag(+) to this mixture does not affect the other metals' distributions, but reflects binding at other protein sites.     

Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints.

ATR, a phosphatidylinositol kinase-related protein homologous to ataxia telangiectasia mutated (ATM), is important for the survival of human cells following many forms of DNA damage. Expression of a kinase-inactive allele of ATR (ATRkd) in human fibroblasts causes increased sensitivity to ionizing radiation (IR), cis-platinum and methyl methanesulfonate, but only slight UV radiation sensitivity. ATRkd overexpression abrogates the G2/M arrest after exposure to IR, and overexpression of wild-type ATR complements the radioresistant DNA synthesis phenotype of cells lacking ATM, suggesting a potential functional overlap between these proteins. ATRkd overexpression also causes increased sensitivity to hydroxyurea that is associated with microtubule-mediated nuclear abnormalities. These observations are consistent with uncoupling of certain mitotic events from the completion of S-phase. Thus, ATR is an important component of multiple DNA damage response pathways and may be involved in the DNA replication (S/M) checkpoint.     

Quantitative fluorescence correlation spectroscopy reveals a 1000-fold increase in lifetime of protein functionality

We have investigated dilute protein solutions with fluorescence correlation spectroscopy (FCS) and have observed that a rapid loss of proteins occurs from solution. It is commonly assumed that such a loss is the result of protein adsorption to interfaces. A protocol was developed in which this mode of protein loss can be prevented. However, FCS on fluorescent protein (enhanced green fluorescent protein, mCherry, and mStrawberry) solutions enclosed by adsorption-protected interfaces still reveals a decrease of the fluorescent protein concentration, while the diffusion time is stable over long periods of time. We interpret this decay as a loss of protein functionality, probably caused by denaturation of the fluorescent proteins. We show that the typical lifetime of protein functionality in highly dilute, approximately single molecule per femtoliter solutions can be extended more than 1000-fold (typically from a few hours to >40 days) by adding compounds with surfactant behavior. No direct interactions between the surfactant and the fluorescent proteins were observed from the diffusion time measured by FCS. A critical surfactant concentration of more than 23 μM was required to achieve the desired protein stabilization for Triton X-100. The surfactant does not interfere with DNA-protein binding, because similar observations were made using DNA-cutting restriction enzymes. We associate the occurrence of denaturation of proteins with the activity of water at the water-protein interface, which was recently proposed in terms of the “water attack model”. Our observations suggest that soluble biomolecules can extend an influence over much larger distances than suggested by their actual volume.     

Quantification of protein concentration by the Bradford method in the presence of pharmaceutical polymers

We investigated how the Bradford assay for measurements of protein released from a drug formulation may be affected by a concomitant release of a pharmaceutical polymer used to formulate the protein delivery device. The main result is that polymer-caused perturbations of the Coomassie dye absorbance at the Bradford monitoring wavelength (595 nm) can be identified and corrected by recording absorption spectra in the region of 350–850 mm. The pharmaceutical polymers Carbopol and chitosan illustrate two potential types of perturbations in the Bradford assay, whereas the third polymer, hydroxypropylmethylcellulose (HPMC), acts as a nonperturbing control. Carbopol increases the apparent absorbance at 595 nm because the polymer aggregates at the low pH of the Bradford protocol, causing a turbidity contribution that can be corrected quantitatively at 595 nm by measuring the sample absorbance at 850 nm outside the dye absorption band. Chitosan is a cationic polymer under Bradford conditions and interacts directly with the anionic Coomassie dye and perturbs its absorption spectrum, including 595 nm. In this case, the Bradford method remains useful if the polymer concentration is known but should be used with caution in release studies where the polymer concentration may vary and needs to be measured independently.     

Electrochemical detection and characterization of proteins

On-line detection of serum proteins is of clinical relevance, in detecting leaks and biofouling in hemofiltration equipment, biofilm growth on prosthetic devices, or hemolysis within a prosthetic or therapeutic device. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to detect and analyze micromolar concentrations of four globular proteins of clinical importance. CV testing showed that identification and quantification of each of these proteins was possible through analysis of current changes at specific potentials. Preliminary CV studies into the contamination of Bovine Serum Albumin with a microgram amount of one of the other three proteins illustrated that direct detection of the contaminant protein was possible. The analysis of the EIS data demonstrated that with increase in relative concentration of proteins, the amount of electroactive proteins adsorption at the interface increases, leading to increase in surface charge density and capacitance, especially for lower molecular weight proteins. The impedance data was used to determine the values of Gibbs adsorption energy, adsorption coefficients for the four proteins, and develop an equivalent circuit model for the protein-containing solutions.