Vladimír Setnička

Analysis of human blood plasma and hen egg white by chiroptical spectroscopic methods (ECD, VCD, ROA)

AbstractChiroptical methods are widely used in structural and conformational analyses of biopolymers. The application of these methods to investigations of biofluids would provide new avenues for the molecular diagnosis of protein-misfolding diseases. In this work, samples of human blood plasma and hen egg white were analyzed using a combination of conventional and chiroptical methods: ultraviolet absorption/electronic circular dichroism (UV/ECD), Fourier transform infrared absorption/vibrational circular dichroism (FTIR/VCD), and Raman scattering/Raman optical activity (Raman/ROA). For comparison, the main components of these substances—human serum albumin (HSA) and ovalbumin (Ova)—were also analyzed by these methods. The ultraviolet region of the ECD spectrum was analyzed using the CDNN CD software package to evaluate the secondary structures of the proteins. The UV/ECD, FTIR/VCD, and Raman/ROA spectra of the substances were quite similar to those of the corresponding major proteins, while some differences were also detected and explained. The conclusions drawn from the FTIR/VCD and Raman/ROA data were in good agreement with the secondary structures calculated from ECD. The results obtained in this work demonstrate that the chiroptical methods used here can be applied to analyze not only pure protein solutions but also more complex systems, such as biological fluids. FigureAnalysis of human blood plasma and hen egg white by ECD, VCD and ROA.

Can Chiroptical Spectroscopy be Used for the Analysis of Blood Plasma

As an improvement on currently used methods of molecular spectroscopy, we used chiroptical techniques (electronic circular dichroism, fluorescence detected circular dichroism, and Raman optical activity [ROA]) to investigate the human blood plasma. To avoid the degradation of plasma samples, we measured them directly without any further preparation. We also tested cutoff weight filters (Amicon Ultra 100, 30, 10, and 3 kDa by Merck Millipore) to reduce undesirable fluorescence in the ROA and Raman spectra and also to remove the most abundant protein in the plasma-human serum albumin. The obtained spectra show that the ultrafiltration has a positive effect on undesirable fluorescence in ROA and Raman and also could reduce the amount of albumin in the plasma. Our results suggest that blood plasma can be successfully measured by the aforementioned methods. Therefore, these methods can potentially be useful for following research in the development of new, noninvasive, and reliable screening methods of clinical diagnostics.

Vibrational and electronic circular dichroism as powerful tools for the conformational analysis of cationic antimicrobial peptides

Antimicrobial and hemolytic activities of cationic α-helical antimicrobial peptides depend on their ability to adopt an amphipathic α-helical conformation on the cell membrane surface. Using vibrational and electronic circular dichroism, we carried out a conformational study of two α-helical antimicrobial peptides, melectin and antapin, in various environments mimicking bacterial and eukaryotic membranes. The results showed a significant difference in the content of α-helical conformation in the environment mimicking the bacterial and eukaryotic membranes.Graphical abstract

Conformational study of melectin and antapin antimicrobial peptides in model membrane environments

Antimicrobial peptides have long been considered as promising compounds against drug-resistant pathogens. In this work, we studied the secondary structure of antimicrobial peptides melectin and antapin using electronic (ECD) and vibrational circular dichroism (VCD) spectroscopies that are sensitive to peptide secondary structures. The results from quantitative ECD spectral evaluation by Dichroweb and CDNN program and from the qualitative evaluation of the VCD spectra were compared. The antimicrobial activity of the selected peptides depends on their ability to adopt an amphipathic α-helical conformation on the surface of the bacterial membrane. Hence, solutions of different zwitterionic and negatively charged liposomes and micelles were used to mimic the eukaryotic and bacterial biological membranes. The results show a significant content of α-helical conformation in the solutions of negatively charged liposomes mimicking the bacterial membrane, thus correlating with the antimicrobial activity of the studied peptides. On the other hand in the solutions of zwitterionic liposomes used as models of the eukaryotic membranes, the fraction of α-helical conformation was lower, which corresponds with their moderate hemolytic activity.

Cocaine Hydrochloride Structure in Solution Revealed by Three Chiroptical Methods

Structure and flexibility of natural compounds determine their biological activity. In the present study, electronic circular dichroism (ECD), vibrational circular dichroism (VCD), and Raman optical activity (ROA) spectra of cocaine hydrochloride in aqueous solutions were measured and related to the structure with the aid of density functional theory (DFT) computations. Additional measurements in deuterated environment made assignment of vibrational bands easier. The results suggest that the prevalent cocaine conformation in solution differs from that adopted in hydrochloride crystal. The spectroscopic results and computational analysis are consistent with X-ray structures of known cocaine-receptor complexes, in which the compound adopts a variety of conformations. All three kinds of chiroptical spectra exhibited significantly greater conformational sensitivity than unpolarized absorption or Raman scattering. The ROA technique provided the largest number of well-resolved bands, bearing rich structural information.

Chiroptical spectroscopy and metabolomics for blood-based sensing of pancreatic cancer

To enable the early diagnosis of pancreatic cancer, the search for and definition of reliable biomarkers remain a subject of great interest, with the specificity and sensitivity of the currently used biomarkers being below the required values. We tested a novel diagnostic approach for pancreatic cancer based on the specific molecular signature of blood plasma components. To acquire more detailed structural information, structure-sensitive chiroptical methods (electronic circular dichroism and Raman optical activity) were supplemented by conventional Raman and infrared spectroscopies. The obtained spectra were subsequently processed by linear discriminant analysis yielding high values of specificity and sensitivity. In addition, to monitor not only large biomolecules as potential biomarkers but also those of low molecular weight, we conducted an analysis of blood plasma samples by using metabolomics. The achieved results suggest a panel of promising biomarkers for a reliable detection of pancreatic cancer.

Structure determination of butylone as a new psychoactive substance using chiroptical and vibrational spectroscopies

Recently, there has been a worldwide substantial increase in the consumption of new psychoactive substances (NPS), compounds that mimic the structure of illicit drugs, such as amphetamines or ecstasy. The producers try to avoid the law by a slight modification of illicit structures, thereby developing dozens of temporarily legal NPS every year. The current trends in the detection and monitoring of such substances demand a fast and reliable analysis. Molecular spectroscopy represents a highly effective tool for the identification of NPS and chiroptical methods can provide further information on their 3D structure, which is the key for the determination of their biological activity. We present the first systematic study of NPS, specifically butylone, combining chiroptical and vibrational spectroscopies with ab initio calculations. According to density functional theory calculations, 6 stable lowest energy conformers of butylone were found and their molecular structure was described. For each conformer, the relative abundance based on the Boltzmann distribution was estimated, their population weighted spectra predicted and compared to the experimental results. Very good agreement between the experimental and the simulated spectra was achieved, which allowed not only the assignment of the absolute configuration, but also a precise description of the molecular structure.

Chiroptical Spectroscopy of Biofluids

Abstract As it was proved many times, the inherent sensitivity to the slightest structural details makes chiroptical spectroscopy a method of choice, when conventional techniques of molecular spectroscopy reach their limits. Taking advantage of such benefit, a smooth analysis of various chiral molecules may be performed, inspecting, for example, active pharmaceutical ingredients, protein conformation, or supramolecular assemblies. The studies of large biomolecules not only in model solutions under strictly controlled experimental conditions, but also in biofluids representing their natural environment have been a challenge to chiroptical spectroscopy. In the last few decades, some research groups were able to overcome the obstacles accompanying biofluid analysis and, thus, took a step toward a deeper insight into the structure–function relationship of biomolecules.

Electronic circular dichroism for the detection of microalbuminuria

Over the past decades, chiroptical spectroscopy has proved its incomparable ability to elucidate the structure and spatial arrangement of chiral molecules. Systematic analysis of biomolecules in the natural environment of biofluids, however, remains challenging. In this study, we used chiroptical spectroscopy to monitor urinary levels of human serum albumin. Not only severe proteinuria but even just a slightly increased urinary excretion of albumin (microalbuminuria) may indicate serious health complications, especially for diabetic individuals. Given the chiral nature of albumin and its typical spectral pattern, it may be easily observable by chiroptical spectroscopy, particularly electronic circular dichroism. The performed chiroptical analysis of urine not only allowed the detection of clinically confirmed microalbuminuria but was also able to reveal this pathological condition in cases beyond the diagnostic capability of common clinical procedures. Thus, our approach suggests that electronic circular dichroism is a useful tool for the fast and reliable qualitative monitoring of microalbuminuria with the potential for a quantitative analysis in the future.

Reply to Comments by Pescitelli and Bruhn on “Cocaine Hydrochloride Structure in Solution Revealed by Three Chiroptical Methods”

We thank G. Pescitelli and T. Bruhn for the comments on our computations. Their Correspondence is well-written and quite appropriate. The authors look more closely at a DFT-related error of the calculations. They find a large effect of the dispersion correction even within one conformer, which is rather surprising for us. We would not agree with some formulations in the text, but this is highly subjective. For example, their spectrum is still off by 10 nm in wavelength scale and the intensity error is 300 %, so that this result does not look “extremely good”. Another thing is whether ECD is more reliable than VOA. Without clear criteria, such as RMS error between calculated and experimental energies/intensities one can argue about this forever. Viewed from certain perspective, the Correspondence itself shows that ECD may be quite problematic. But the authors are certainly right that the techniques are complementary and one should not dig arbitrary ditches between them.