Investigation of the Binding Behavior between the S-heterocyclic Aromatic Palladium(II) Complex and Human Serum Albumin: Spectroscopic Approach

The interaction of 1, 10-phenanthroline octhyldithiocarbamato palladium(II) nitrate ([Pd(Oct-dtc)(phen)]NO₃) with human serum albumin (HSA) has been investigated by various spectroscopic techniques under physiological conditions. Here, HSA was titrated with the Pd(II) complex, followed by UV–Vis absorption spectroscopy to estimate a binding constant (K_b) and other thermodynamic parameters. The results indicate that the Pd (II) complex has a high affinity for bind HSA. Thermodynamic analysis showed that the enthalpy (ΔH°) and entropy changes (ΔS°) are positive and Gibbs free energy change (ΔG°) is negative which indicated that hydrophobic interactions played the predominant role in the binding process. Fluorescence spectroscopy were used to show the mechanism and binding parameters of this interaction. Utilizing the Stern–Volmer equation, the Pd(II) complex quenched the intrinsic fluorescence of HSA via a static quenching procedure. The specific binding distances between the tryptophan (donor) proteins and Pd(II) complex (acceptor) were estimated by Forster resonance energy transfer. The CD results also showed the conformational changes on serum albumin upon binding with the Pd(II) complex.     

Efficient adsorptive extraction materials by surface protein-imprinted polymer over silica gel for selective recognition/separation of human serum albumin from urine

In this study, we report the development of adsorptive extraction materials by surface protein-imprinted polymers (MIPs) over silica gel for selective recognition/separation of human serum albumin (HSA) from urine. The HSA-imprinted polymers prepared on silica particle had at interface between the silica gel and different MIPs greatly produced enrichment for the binding of protein from the urine. The solid-phase extraction of the optimized polymer layer was prepared by copolymerization of methacrylic acid (MAA), acrylamide (AAm), and dimethylaminoethylmethacrylate (DMAEMA) and a crosslinker methylenebisacrylamide (MBA) at the mole ratio of 1:158:88 (T:M:C) and showed moderate affinity (<10⁴ order M⁻¹) toward target protein HSA and selectivity. Four analogues, egg white albumin (EWA), bovine serum albumin (BSA), lysozyme (Lyz), and creatinine (Cre) were selected to study the binding efficiency of MIPs in single and binary protein solutions. We studied the influence on recognition ability for HSA and found that prepolymer mixture and matrix flexibility of the optimized thin polymer layer (35 ± 10 nm) on the submicrosilica particles. The high-binding affinity (Q_MIP, 86.7 mg g⁻¹) and fast kinetics (180 min) were observed for this synthesized HSA-MIP when compared with other reported HSA-MIPs in surface imprinting (5.9 and 11.3 mg g⁻¹) and epitope surface imprinting (46.6 mg g⁻¹) methods. We demonstrated the application in real and synthetic urine samples that the approach allowed the efficient adsorption of HSA in real urine (129.48 mg g⁻¹) is almost double to the binding of HSA in synthetic urine (67.84 mg g⁻¹). Apart from this, only minor interference of Cre (2.74 mg g⁻¹) was observed, eventhough Cre is the final metabolite in urine. These adsorptive submicrosilica materials have potential in the pharmaceutical industry and clinical analysis applications. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 46894.     

Blood parameters of young calves at abattoirs are related to distance transported and farm of origin

Nonreplacement dairy calves, or bobby calves, are fasted and transported to abattoirs from as young as 5 d of age in Australia. The aims of this cross-sectional observational study were (1) to assess the welfare status, as measured by blood parameters, of bobby calves in the commercial supply chain after transport and lairage, and (2) to assess whether distance and duration of transport are risk factors for poor bobby calf welfare, as measured by blood parameters. We hypothesized that bobby calves transported greater distances would be more likely to show evidence of compromised welfare, as measured by blood indicators of hydration, energy status, and muscle fatigue or damage. We also hypothesized that there would be a large amount of variability in indicators of energy status between calves from different farms. We analyzed blood samples collected at slaughter over a spring and an autumn calving period from 4,484 Australian bobby calves aged approximately 5 to 14 d old from 3 different states, after transport, fasting, and lairage. Packed cell volume (PCV), plasma glucose, and serum urea, total protein, β-hydroxybutyrate (BHB), and creatine kinase (CK) were measured. Radio frequency identification ear tag data were used to estimate the distance that the calves were transported and to identify the farm of origin. Data were analyzed using linear mixed models, except for BHB, which was analyzed using a Goodman-Kruskal gamma test due to left censoring of the data. Twelve percent of calves showed evidence of anemia (PCV less than 0.23 L/L), and 11% had urea concentrations consistent with dehydration (urea more than 7.7 mmol/L). Thirty-six percent of calves had CK activity above normal resting values, and 1% of calves had CK >2,000 U/L, indicating muscle fatigue or damage. Distance transported had significant effects on all blood variables except urea and BHB. With increasing distance transported, calves were more likely to show evidence of a negative energy balance (low plasma glucose) or dehydration (high PCV or total protein). The estimated effect of distance overall was small, but for calves transported more than 500 km, plasma glucose concentration declined more per kilometer. The calves' farm of origin accounted for a reasonable amount of the random variation between calves for plasma glucose (20%). Our results suggest that longer transport distances may increase the risk of poor calf welfare (dehydration, negative energy balance) after transport, and on-farm calf management (e.g., nutrition, timing of feeding before transport) may affect transported calves' energy status; improving this area could result in better energy availability during fasting.     

Interactions of different polyphenols with bovine serum albumin using fluorescence quenching and molecular docking

Polyphenols are responsible for the major organoleptic characteristics of plant-derived foods and beverages. Here, we investigated the binding of several polyphenols to bovine serum albumin (BSA) at pH 7.5 and 25 °C: catechins [(−)-epigallocatechin-3-gallate, (−)-epigallocatechin, (−)-epicatechin-3-gallate], flavones (kaempferol, kaempferol-3-glucoside, quercetin, naringenin) and hydroxycinnamic acids (rosmarinic acid, caffeic acid, p-coumaric acid). Fluorescence emission spectrometry and molecular docking were applied to compare experimentally determined binding parameters with molecular modelling. Among these polyphenols, (−)-epicatechin-3-gallate showed the highest Stern–Volmer modified quenching constant, followed by (−)-epigallocatechin-3-gallate. Similarly, (−)-epicatechin-3-gallate had the highest effect on the Circular Dichroic spectrum of BSA, while the changes induced by other polyphenols were negligible. Molecular docking predicted high binding energies for (−)-epicatechin-3-gallate and (−)-epigallocatechin-3-gallate for the binding site on BSA near Trp213. Our data reveal that the polyphenol structures significantly affect the binding process: the binding affinity generally decreases with glycosylation and reduced numbers of hydroxyl groups on the second aromatic ring.     

Antisolvent micronization of BSA using supercritical mixtures carbon dioxide + organic solvent

In this work, expanded liquid antisolvent (ELAS) process has been used to micronize bovine serum albumin (BSA) solubilized in water. Carbon dioxide mixtures with ethanol, acetone or isopropyl alcohol, at expanded liquid conditions, have been used as the antisolvent. The effect of process parameters, such as the kind of co-antisolvent and the organic co-antisolvent/water/carbon dioxide mole fraction on the morphology and dimensions of the precipitates, was studied. Changing co-antisolvent and operating conditions, we obtained nanoparticles (with a mean diameter of about 60 nm ± 10 nm), sub-microparticles (with a mean diameter of 470 nm ± 130 nm), microparticles (with a mean diameter of 0.93 μm ± 0.37 μm) and expanded microparticles with an empty core (with a mean diameter of about 9 μm ± 5 μm). Fourier transform infrared analysis on BSA powders revealed that, using acetone as co-antisolvent, no modifications of the protein secondary structure were induced by ELAS processing.     

Mass spectrometry of peptides and proteins from human blood

It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post‐translational modifications. However, blood is a complex mixture that may contain many proteins first expressed in cells and tissues. The complete analysis of blood proteins is a daunting task that will rely on a wide range of disciplines from physics, chemistry, biochemistry, genetics, electromagnetic instrumentation, mathematics and computation. Therefore the comprehensive discovery and analysis of blood proteins will rank among the great technical challenges and require the cumulative sum of many of mankind's scientific achievements together. A variety of methods have been used to fractionate, analyze and identify proteins from blood, each yielding a small piece of the whole and throwing the great size of the task into sharp relief. The approaches attempted to date clearly indicate that enumerating the proteins and peptides of blood can be accomplished. There is no doubt that the mass spectrometry of blood will be crucial to the discovery and analysis of proteins, enzyme activities, and post‐translational processes that underlay the mechanisms of disease. At present both discovery and quantification of proteins from blood are commonly reaching sensitivities of ～1 ng/mL. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:685–732, 2011     

A reliable and sensitive time-resolved fluorescent immunochromatographic assay (TRFICA) for ochratoxin A in agro-products

Immunochromatographic assays (ICAs) are considered as a suitable diagnostic tool for the detection of mycotoxins. Mycotoxins and especially, ochratoxin A are analytes with more demanding sensitivity requirements. To enhance the sensitivity of current immunochromatographic assays for ochratoxin A (OTA), a novel sensitive ICA was developed in this study. In the assay, microspheres enclosing fluorescent europium (III) [Eu(III)] nanoparticles (EuNPs) were used as a label for OTA monoclonal antibody (OTA-mAb) conjugation. Accordingly, assay was called time-resolved fluorescent immunochromatographic assay (TRFICA). The test strip was composed of three parts: a sample pad, nitrocellulose membrane and an absorbent pad. As for detection, a proper concentration of conjugated microspheres was pipetted into the microtube and sample extract was added to it. Then the strip was inserted into the tube and the fluid flow along the strip. The TRFICA results were obtained in 8 min and read by a portable TRFICA strip reader. The established method allows quantitative determination of OTA with limit of detection as low as 1.0 μg kg⁻¹ in the samples. For validation, spiked samples including wheat, maize, soybean and rice were respectively assayed by TRFICA and a standard high performance liquid chromatography equipped with a fluorescence detector (HPLC-FLD), and good agreement of results was obtained between two methods.