Characterization of Soybean Protein Isolate-Food Polyphenol Interaction via Virtual Screening and Experimental Studies

(1) Background: Protein–polyphenol interactions have been widely studied regarding their influence on the properties of both protein and the ligands. As an important protein material in the food industry, soybean protein isolate (SPI) experiences interesting changes through polyphenols binding. (2) Methods: In this study, a molecular docking and virtual screening method was established to evaluate the SPI–polyphenol interaction. A compound library composed of 33 commonly found food source polyphenols was used in virtual screening. The binding capacity of top-ranking polyphenols (rutin, procyanidin, cyanidin chloride, quercetin) was validated and compared by fluorescence assays. (3) Results: Four out of five top-ranking polyphenols in virtual screening were flavonoids, while phenolic acids exhibit low binding capacity. Hydrogen bonding and hydrophobic interactions were found to be dominant interactions involved in soybean protein–polyphenol binding. Cyanidin chloride exhibited the highest apparent binding constant (Ka), which was followed by quercetin, procyanidin, and rutin. Unlike others, procyanidin addition perturbed a red shift of SPI fluorescence, indicating a slight conformational change of SPI. (4) Conclusions: These results suggest that the pattern of SPI–polyphenol interaction is highly dependent on the detailed structure of polyphenols, which have important implications in uncovering the binding mechanism of SPI–polyphenol interaction.

Increase in the apparent intercalation ability of a platinum complex via multivalency by installation into the sidechain of a graft copolymer and observation of structural changes in the intercalated DNA

Pt complexes increase their apparent binding constant by grafting on sidechains of polymer segments via multivalent effect.

Quartz Crystal Microbalance (QCM) Piezo-Immunosensors: A Novel Technique to Study CD20 Antigen Interaction with Rituximab and Its Clinical Implications

Background: CD20 antigen, expressed on greater than 90% of B-cell lymphomas, is an established target for antibody therapy with monoclonal antibodies like Rituximab, which can selectively deplete CD20-expressing cells in peripheral blood and lymphoid tissues. Understanding properties and interactions of CD20 antigens is quintessential for developing better targeted agents and overcoming resistance. It has not been clearly established yet whether CD20 antigen density corresponds with response to Rituximab. Flow cytometry is still the most widely used technique to detect CD20 level in human serum which is expensive, time consuming and does not reveal any details of interaction between the molecules. We have developed a new innovative biosensor based novel technique to study real time interaction of CD20 antigens with Rituximab using QCM Piezo-immunosensor. This quantitative label free peptide based assay can be used to characterize cell surface antigen, to study antigen- antibody interactions and obtain understanding of mechanisms of resistance to therapy. Method: The immobilization of Raji cells on the QCM gold electrode surface using RGD tripeptide was electrochemically confirmed (Figure 1). The real-time processes of attachment of Raji cells on the gold electrode and the subsequent binding of Rituximab to the cells were studied using QCM biosensor. The interaction between Rituximab and Raji cells led to the increased resonant frequency shifts (df0) in the studied antibody concentration range from 5 to 250 μg mL-1 following the Langmuir adsorption model (Figure 2). From these observations, the apparent binding constant between a single-layer of Rituximab and Raji cells was calculated to be 1.6×106 M-1 (Figure 3). Control experiments using other therapeutic antibodies (i.e., Trastuzumab and Bevacizumab) and different cells (i.e., T cells and endothelial cells) proved very specific interaction between Rituximab and CD20 antigens on B cells. Calcium and Manganese ions were added to the cell culture and corresponding responses by QCM were monitored. Results: CD20 binding with Rituximab was very specific. This binding decreased the electrochemical activity and stability of the cells, supporting the cell lysis mechanisms of action of Rituximab. We have shown a systematic approach for using QCM technique to quantify the apparent binding constant between Raji cells and Rituximab which can reveal CD20 antigen density. Moreover, increased QCM responses were found in the presence of Ca2+ ions and high concentration Mn2+ ions, supporting the function of CD20 as a calcium ion channel. Microscopic inspection proved that increased Ca2+ ions could promote the Rituximab binding and cell lysis induced by Rituximab, which was not seen with Manganese. Conclusion: CD20 antigen density and interactions of CD20 antigens with respective monoclonal antibodies will help physicians to determine the clinical efficacy and resistance mechanisms to targeted antibodies like Rituximab and Ofatumumab. For the first time, we have established a low cost, highly sensitive, fast, synthetic, QCM assay which could be used as a basis for developing a new generation of affinity-based Immunosensor assays. This real time capability of QCM and its simplicity of operation are highly suitable for multipurpose studies on living cells including cell immobilization, cytotoxicity of drugs, and the cell action mechanisms. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

The Investigation of the Interaction between Daidzin and Bovine Serum Albumin by Fluorescence Spectroscopy

We investigated the mutual interaction of daidzin with bovine serum albumin (BSA) by fluorescence spectroscopy. The results revealed that daidzin cause the fluorescence quenching of BSA through a static quenching procedure. The Stern-Volmer quenching constant (Ksv) were calculated at different temperature. The binding site (n), apparent binding constant (Ka) and corresponding thermodynamic parameters △Go, △Ho, △Sowere calculated and the van der Waals interaction, hydrogen bonds and hydrophobic interactions play an important role in stabilizing the complex. Besides, we also studied the effect of Cu2+, Ni2+, Mn2+and Co2+on the binding constants between daidzin and BSA, it is shows that the binding of BSA and daidzin is strengthened in the presence metal ions.

Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, <em>Schizosaccharomyces pombe</em> Pot1 and <em>Saccharomyces cerevisiae </em>Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

Interaction Mechanism between Bovine Serum Albumin and Polyethylene Glycol Revealed with Elastic Light Scattering Spectroscopy

In order to gain deeper insight into the interaction mechanism between bovine serum albumin (BSA) and polyethylene glycol (PEG), the present work applied elastic light scattering (ELS) spectroscopy to investigate the interaction between BSA and PEG, and explore the effects of concentration and molecular weight of PEG on the interaction at physiological pH. The results showed that the interaction force existed between linear PEG and spherical BSA molecules was mainly hydrogen bonding. In addition, the apparent binding constant of system was evaluated by model calculation.

Characterizing the binding of nucleotide ATP on serum albumin by 31P NMR diffusion

The pulsed-field-gradient (PFG) 31P NMR diffusion spectra were measured under varied sample conditions to characterize the low-affinity binding of adenosine 5′-triphosphate (ATP) on human serum albumin (HSA) or bovine serum albumin (BSA). The NMR diffusion constants of ATP, ATP–HSA, or ATP–BSA were illustrated as function of ATP concentrations. The binding curves of ATP–HSA and ATP–BSA were identical but strikingly different from the ATP curve. Using a “Scatchard plot”, the apparent binding constant (K) and number of ATP binding sites (n) on serum albumin were evaluated as K = 75.25 (mol/L)–1 and n = 10, respectively. At a pH < 5.0 and a pH > 9.0 or a temperature > 45 °C, the diffusion data of ATP–HSA were found to increase remarkably, suggesting that the dissociation of ATP from HSA was largely enhanced, probably because of pH- or heat-induced protein structural change, degradation, or aggregation. In addition, our data indicated that ADP was strongly competitive with ATP for the low-affinity binding to HSA, but heptanone and Cl– were essentially noncompetitive. These results are important for further elucidating the interaction of ATP with serum albumin and its possible effect on related bioprocesses. The method can be well applied to study the binding of other nucleotides/nucleosides on proteins.

Spectroscopic and Electrochemical Studies on the Interaction of Epirubicin with Fish Sperm DNA

The interactions between epirubicin (EPR) and double stranded DNA (ds-DNA) have been studied by UV-Vis spectrophotometry, cyclic voltammetry, fluorescence spectroscopy and viscometery. The apparent binding constant of epirubicin with DNA was found to be 3.8 × 105mol-1L and was studied at different temperatures. It indicated that the former method could be successfully applied to the determination of epirubicin. Also, the voltammetric behavior of EPR was investigated at glassy carbon electrode using cyclic voltammetry. Thermodynamic parameters including ∆H0, ∆G0and ∆S0were determined as -5.7×104, -3.13×104J mol-1and -87.96 J mol-1K-1 respectively. One indication of the strong interaction between DNA and EP was increasing of viscosity. The diffusion coefficients of EP in the absence (D0)fand presence of ds-DNA (D0)bwas calculated as 5.04×10-6and 1.13×10-6 Cm2s-1respectively. According to the thermodynamic parameters, the main binding force could be judged. The experimental results revealed that EPR and ds-DNA had strong interactions. The mechanism of quenching belonged to static quenching and the main sort of binding force was intercalation.