Imidazole Buffer Research Articles

Purification of pineapple bromelain by IMAC chromatography using chlorophyll-activated macroporous matrices

This study investigated the purification of bromelain obtained from pineapple fruit using a new adsorbent for immobilized metal ion affinity chromatography (IMAC), with chlorophyll obtained from plant leaves as a chelating agent. The purification of bromelain was evaluated in batches from the crude extract of pineapple pulp (EXT), and the extract precipitated with 50 % ammonium sulfate (EXT.PR), the imidazole buffer (200 mM, pH 7.2) being analyzed and sodium acetate buffer, pH 5.0 + 1.0 NaCl as elution solutions. All methods tested could separate forms of bromelain with molecular weights between ±21 to 25 kDa. Although the technique using EXT.PR stood out in terms of purity, presenting a purification factor of around 3.09 ± 0.31 for elution with imidazole and 4.23 ± 0.12 for acetate buffer solution. In contrast, the EXT methods obtained values between 2.44 ± 0.23 and 3.21 ± 0.74 for elution with imidazole and acetate buffer, respectively, for purification from EXT.PR has lower yield values (around 5 %) than EXT (around 15 %). The number of steps tends to reduce yield and increase process costs, so the purification process in a monolithic bed coupled to the chromatographic system using the crude extract was evaluated. The final product obtained had a purification factor of 6, with a specific enzymatic activity of 59.61 ± 0.00 U·mg−1 and a yield of around 39 %, with only one band observed in the SDS-PAGE electrophoresis analysis, indicating that the matrix produced can separate specific proteins from the total fraction in the raw material. The IMAC matrix immobilized with chlorophyll proved promising and viable for application in protease purification processes.

Purification and characterization of human glycerol 3-phosphate dehydrogenases (mitochondrial and cytosolic) by NAD+/NADH redox method

Glycerol 3-phosphate (G3P) shuttle is composed of mGPDH and cGPDH and serves as the interface between carbohydrate- and lipid-metabolism. Recently, these metabolic enzymes have been implicated in type II diabetes mellitus but the detailed kinetic parameters and crystal structure of human mGPDH is unknown, though fewer studies on cGPDH are available. To characterize these enzymes, the human mGPDH and cGPDH genes were optimized and cloned into the pET-SUMO vector and pET-24a(+) vector, respectively, and over-expressed in Escherichia coli BL21 (DE3). However, SUMO-mGPDH was expressed as inclusion bodies. Hence, various culture parameters, solubilizing agents and expression vectors were used to solubilize the protein but they did not produce functional SUMO-mGPDH. Over-expression of SUMO-mGPDH along with molecular chaperone (pG-KJE8) produced a functional SUMO-mGPDH. The functional SUMO-mGPDH was purified and characterized using NAD+/NADH redox method. cGPDH was also over-expressed and purified for its characterization. DLS analysis and CD spectra of the purified proteins were performed. The mGPDH was a monomeric enzyme with MW of ∼74 kDa and displayed optimal activity in the Tris-HCl buffer (pH 7.4); while, cGPDH was a homodimer with a monomeric MW of ∼37 kDa and showed optimal activity in imidazole buffer (pH 8.0). The Kmapp was 0.475 mM for G3P, and 0.734 mM for DHAP. These methods may be used to characterize these enzymes to understand their role in metabolic disorders.

Investigating the effect of diluents and fat globules on the size measurement of casein micelles by dynamic light scattering

The hydrodynamic size of casein micelles was examined using dynamic light scattering (DLS). Four types of diluents [ultrafiltration (UF) permeate, lactose-free simulated milk ultrafiltrate (SMUF), calcium imidazole buffer, and deionised water] were compared in their effect on the stability of casein micelles. Results of dilution series and kinetic size measurements revealed that both UF permeate and lactose-free SMUF enable size measurements that are hardly affected by the required dilution step, whereas a calcium imidazole buffer containing 10 mm CaCl2 only could provide accurate sizing results within a short time after dilution. The contribution of residual fat globules to the particle size distribution was negligible in skim milk. For semi-skimmed milk, the size of the casein micelles could be estimated by subtracting the contribution of the fat droplets (obtained upon dilution in a casein dissolving solution) from the raw DLS data (as obtained upon dilution in milk ultrafiltrate).

Cover Feature: Effects of First‐Coordination Sphere and Buffers on the Nitrosyl‐Nitrite Conversion Rate in Ru(II) Complexes (Eur. J. Inorg. Chem. 30/2022)

The Cover Feature shows comparative kinetic studies of nitrosyl ruthenium complexes such as cis-[Ru(NO)(bpy)2L]3+ (L=4-pic, py, isn, and 4-acpy) used as a platform for understanding the effect of the interaction between biphthalate, phosphate, and imidazole buffer solutions and the ruthenium complex to enable the conversion of nitrosyl to nitrite at pH=6.5. The buffer can dramatically improve the rate of the reaction to make it around three times faster in the order: biphthalate<phosphate<imidazole. The reaction rate was also dependent on the π-acceptor character of ligand “L” in cis-[Ru(NO)(bpy)2L]3+, with kobs increasing in the following order: 4-pic<py<isn< 4-acpy. All the kinetic results based on the interaction mechanism are supported by the theoretical calculations. More information can be found in the Research Article by R. Santana da Silva and co-workers.

Effects of First‐Coordination Sphere and Buffers on the Nitrosyl‐Nitrite Conversion Rate in Ru(II) Complexes

AbstractNitrogen oxide derivative ruthenium compounds can deliver nitric oxide (NO) by chemical, electrochemical, or photochemical processes. Among these compounds, nitrosyl ruthenium complexes have been one of the most studied species. The σ‐ and π‐character of the co‐ligand L in [RuL5(NO)]n+ exerts a unique and determining effect on the structure of the complex and the NO delivery mode. We have found inconsistencies in this assertion. How the buffer medium influences the hydrolysis rate constant of nitrosyl ruthenium complexes such as cis‐[Ru(NO)(bpy)2L]3++(L=4‐pic; py; isn and 4‐acpy) has been evaluated in biphthalate, phosphate, and imidazole buffer solutions. For the studied ruthenium complexes, hydrolysis depended on the π‐acceptor character of ligand “L”, with kobs increasing in the following order: 4‐pic&lt;py&lt;isn&lt;4‐acpy. For nitrite conversion, the relative efficiency of the buffers increased in the order: biphthalate&lt;phosphate&lt;imidazole for all the studied nitrosyl ruthenium complexes. On the basis of these findings, we hypothesized that the second‐coordination sphere interacts with the ruthenium complexes. By conducting theoretical modeling studies, we have elucidated the potential contribution of this interaction to nitrosyl/nitrite interconversion.

A New Reaction for Improved Calibration of EPR Rapid-Freeze Quench Times: Kinetics of Ethylene Diamine Tetraacetate (EDTA) Transfer from Calcium(II) to Copper(II).

The kinetics of the transfer of the chelate, ethylenediamine tetraacetate (EDTA), from Calcium(II) to Copper(II) in imidazole (Im) buffers near neutral pH, corresponding to the conversion, [Cu(II)Im4]2+→ [Cu(II)EDTA]2-, are characterized with stopped-flow absorption spectroscopy and implemented as a tool for calibrating the interval between mixing and freezing, the freeze-quench time (t Q ), of a rapid freeze-quench (RFQ) apparatus. The kinetics of this reaction are characterized by monitoring changes in UV-visible spectra (300 nm) due to changes in the charge-transfer band associated with the Cu2+ ions upon EDTA binding. Stopped-flow measurements show that the rates of conversion of the Cu2+ ions exhibit exponential kinetics on millisecond time scales at pH values less than 6.8. In parallel, we have developed a simple but precise method to quantitate the speciation of frozen solution mixtures of [Cu(II)(EDTA)]2- and tetraimidazole Cu(II) ([Cu(Im)4]2+) in X-band EPR spectra. The results are implemented in a simple high-precision 'recipe' for determining t Q . These procedures are more accurate and precise than the venerable reaction of aquometmyoglobin with azide for calibrating RFQ apparatus, with the benefit of avoiding high-concentrations of toxic azide solutions.

Determining the Effect of pH on Iron Oxidation Kinetics in Aquatic Environments: Exploring a Fundamental Chemical Reaction To Grasp the Significant Ecosystem Implications of Iron Bioavailability

Understanding the controls of the oxidation rate of iron (Fe) in oxygenated aquatic systems is fundamental for students of the Earth and Environmental Sciences as it defines the bioavailability of Fe, a trace metal essential for life. The laboratory experiment presented here was successfully developed and used during a third-year undergraduate lab course at Imperial College London for several years. It employs ultraviolet–visible (UV–vis) spectroscopy calibrated externally with 0 to 50 μM Fe2+ standards created in a 492 μM ferrozine and 0.43 M acetate matrix. The students conducted the oxidation experiments in stirred batch reactors at equilibrium with atmospheric oxygen. The solution contained 40.5 μM initial Fe2+ concentration and a 5.1 mM imidazole buffer. The pH was adjusted to values between 7.22 and 7.77. The students observed a pseudo-first-order reaction with respect to Fe2+ concentration. Plotting the logarithms of the apparent rate constants (k′) at different pH values leads to a gradient of 2.2 ± 0.2 min–1 pH–1, indicating a second-order reaction with respect to OH– concentration, in agreement with published literature. The oxidation reaction occurred rapidly (tens of seconds to tens of minutes) indicating that in oxygenated aquatic systems, Fe3+ will be the dominant oxidation state, significantly reducing the bioavailability of Fe. The simple laboratory experiment presented here allows the students to learn about kinetic parameters for a fundamental chemical reaction. It allows the students to explore the significant implications this has for aquatic ecosystems.

Contribution of the cold shock protein CspA to virulence in Xanthomonas oryzae pv. oryzae.

Summary Xanthomonas oryzae pv. oryzae (Xoo) causes a damaging bacterial leaf blight disease in rice. Cold shock proteins (Csps) are highly conserved nucleic acid‐binding proteins present in various bacterial genera, but relatively little is known about their functions in Xanthomonas. Herein, we identified four Csps (CspA–CspD) in the Xoo PXO99A strain. Deletion of cspA decreased cold adaptation and a few known pathogenic factors, including bacterial pathogenicity, biofilm formation and polysaccharide production. Furthermore, we performed transcriptomic and chromosome immunoprecipitation (ChIP) experiments to identify direct targets of CspA and to determine its DNA‐binding sequence. Integrative data analysis revealed that CspA directly regulates two genes, PXO_RS11830 and PXO_RS01060, by binding to a conserved CCAAT sequence in the promoter region. We generated single‐deletion mutants of each gene and the results indicate that both are responsible for Xanthomonas pathogenicity. In addition, quantitative real‐time polymerase chain reaction and western blotting showed that CspA suppressed the expression of its direct targets. In summary, our study clarifies the characteristics of Csps in Xanthomonas and greatly advances our understanding of the mechanisms underlying the contribution of CspA to bacterial virulence.

Self-association of bovine β-casein as influenced by calcium chloride, buffer type and temperature

The aim of this study was to investigate the aggregation behaviour of a pure β-casein (β-CNpure) and a β-casein concentrate (β-CNconc) as a function of temperature, buffer type (pH 6.8) and the presence of CaCl2. The particle size distribution and turbidity of β-casein (β-CN) dispersions were measured by dynamic light-scattering (DLS) and UV/vis spectroscopy between 4 and 55 °C. Upon heating (4–55 °C), the particle size of both β-CN samples increased, indicating self-association via hydrophobic interactions. It was shown that the self-association of β-CN increased with increasing β-CN concentration and that β-CNpure self-associated at significantly lower concentration than β-CNconc. Both turbidity and particle size measurements showed that the β-CN samples had similar aggregation behaviour in water and imidazole buffer (pH 6.8) but differed in sodium phosphate buffer (pH 6.8), especially at higher ionic calcium concentrations. Fourier Transform Infrared (FTIR) spectroscopy revealed very little change in the secondary structure of β-CN during heating (4–55 °C). The microstructure of β-CN aggregates was monitored during heating from 10 to 55 °C, followed by cooling to 10 °C, using polarised light microscopy. Spherical and heterogeneous aggregates were observed when heated at temperatures above 37 °C, which were reversible upon cooling. This study confirmed that β-CN undergoes self-association on heating that reverses upon cooling, with the aggregation process being highly dependent on the purity of β-CN, the solvent type and the presence of ionic calcium.

In-vitro assessment of the effect of dabigatran on thrombosis of adult and neonatal plasma: comparisons using thromboelastography and microscopic visualization of fibrin clot structure.

: Thromboelastography (TEG) is a global assay used for evaluating features of clot formation in vitro. Dabigatran is a reversible direct inhibitor of thrombin that has not been studied in neonates using a sophisticated global assay, such as TEG. Neonatal hemostasis differs from adult hemostasis in both quantitative and qualitative characteristics. Our aim was to compare the TEG clotting profile of neonatal and adult platelet-poor plasma when exposed to different concentrations of dabigatran. We used commercially collected adult pooled plasma and neonatal cord blood collected from placentas of healthy full term newborns. Platelet-poor plasma was isolated, pooled, and frozen. Prior to experiment, plasma was thawed and filtered. A reaction mixture of CaCl2, corn trypsin inhibitor, tissue factor, and dabigatran in imidazole buffer was mixed with plasma in a TEG cup. Time to clot initiation (R-time), speed of clot strengthening (α-angle), and maximum clot strength (maximal amplitude) were measured. Scanning electron microscopy was performed to evaluate fibrin clot structure. Without dabigatran, there was no significant difference in TEG measurements between neonatal and adult samples. However, neonatal plasma clotting with dabigatran had slower onset, slower speed, and weaker clots that were more porous with thicker fibers, compared with adult plasma clotting. Thus, neonatal plasma may be more sensitive to dabigatran as assessed by our in-vitro TEG study.

The preparation and evaluation of the quality control materials for detection of platelet membrane glycoproteins by flow cytometry

Objective: To prepare the quality control material for detection of platelet membrane glycoproteins by flowcytometry and evaluate the appearance traits, homogeneity and stability of it. Methods: Fresh platelets from the blood group O donors were fixed by the certain concentration of aldehyde solution and then washed by the imidazole buffer. After that, adding certain concentration of lyophilized protection solution into the preparations. The preparations were dispensed to be lyophilized and then were kept refrigerated in 2-8 ℃.According to the protocol of control of lyophilized biological products, the quality indicator for monitoring the prepared process, containing the appearance traits, the residual water, the platelet recovery and the rehydration quality were evaluated. The homogeneity and stability of these preparations were evaluated according to the CNAS-GL03 Guidance on evaluating the homogeneity and stability of samples used for proficiency testing and the ISO Guide 35 Reference material-general and statistical principles for certification. Results: The appearance traits and the rehydration quality of the quality control materials meeted the requirements, with the residual water distributed between 3.96% to 4.04% and the platelet recovery rate ranged from 68% to 72%.The homogeneity evaluation showed that there was no significant difference among the groups(P>0.05). The stability test indicated that the positive rate of platelet membrane glycoproteins CD42b, CD41 and CD62P of the quality control material was -0.14%, -0.14% and 0.74%, respectively, at 16 weeks after storage. There was no linear trend between the percentage of positive platelets with membrane glycoproteins and time(P>0.05). Conclusions: The quality control material for detection of platelet membrane glycoproteins by flow cytometry prepared by us meets the needs of the appearance traits, the residual water, the rehydration quality, the homogeneity and the longtime stability.It is hopeful to be used as internal quality control of the assay in clinic laboratory, the external quality assessment and proficiency evaluation.

Thermal Aggregation of Bovine Serum Albumin in Conventional Buffers: An Insight into Molecular Level Interactions

We have studied the effect of some conventional buffers, which are used frequently for biological research, on the thermal aggregation behavior of bovine serum albumin (BSA). The aggregation kinetics of BSA in buffer solutions were investigated by using UV–Vis spectroscopy. The buffers include sodium phosphate buffer, TRIS buffer and imidazole buffer at physiological pH (7.4). Dynamic light scattering and scanning electron microscopy have been employed to illustrate the size and morphology of protein aggregates. The molecular level interactions of buffer molecules with BSA was probed by various spectroscopic techniques, including UV–Vis, fluorescence, and circular dichroism. The results of this study reveal that the strong interactions of the buffers with protein’s folded/unfolded structures lead to stabilization/destabilization of BSA. We have also explored the possible binding sites of BSA for these buffers using a molecular docking technique.

Optical measurement of the pH value of intransparent samples under high pressure

We present a method to optically study the pH value of intransparent media under pressure. To this end, we prepare and use immobilized pH-value indicators. The indicators are brought into contact to the medium and their optical signal is read out in reflection with a spectrometer. The pH value and pressure response of the indicator were calibrated with imidazole buffers. As an example for its applicability to foodstuff, we study the pressure-induced pH-value change of fat and meat pastes.

Mesoporous silica nanoparticles with different pore sizes for delivery of pH‐sensitive gallic acid

ABSTRACTIn this study, mesoporous silica nanoparticles (MSN) with different pore diameters were used for encapsulation of gallic acid (GA). All nanoparticles were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, N2 adsorption isotherms, scanning electron microscopy, X‐ray diffraction, and z‐potential analysis for determination of their characterizations. The effects of different pore sizes of nanoparticles on loading of GA and its release from GA‐loaded nanoparticles into simulated gastric fluid (SGF, pH 1.2), simulated intestinal and colon fluids (SIF and SCF, pHs 6.8 and 7.4, respectively), simulated body fluid (SBF, pH 7.4), and simulated gastrointestinal tract fluid (SGITF) were investigated. The pore size increased with the increase of mixing time of mesitylene as a swelling agent. GA is an unstable molecule that decomposes in alkaline pH. The imidazole buffer is the best for investigation of GA release into the alkaline media. The increase of nanoparticles pore size made the enhancement of GA loading. The released GA from GA‐loaded nanoparticles into SGF was slower than that into SIF and SBF. Release of GA was strongly pH dependent on the selected media. The antioxidant activity, based on the 1,1‐diphenyl‐2‐picrylhydrazyl assay, of released GA into SGITF and SBF was conserved and correlated strongly with its content in the selected media. © 2014 Curtin University of Technology and John Wiley &amp; Sons, Ltd.

Does the presence of von Willebrand factor in FVIII-deficient plasma influences the measurement of FVIII inhibitor titres in haemophilia A patients?

Reliable measurement of FVIII inhibitor is critical in the follow-up of haemophilia A patients. We performed a multicentre study to evaluate whether the presence of von Willebrand factor (VWF) in FVIII-deficient plasma (FVIII-DP) influences FVIII inhibitor titres. Six French haematology laboratories participated in this study. Three samples with varying FVIII inhibitor titres (1, 5 and 15BU/mL) and one sample without any detectable FVIII inhibitor were tested using four different procedures for FVIII inhibitor assay. The Nijmegen method and a modified assay with imidazole were performed using FVIII-DP with and without VWF in the control mixture and as substrate plasma in the FVIII one stage assay (OSA). Each mixture (reference and test) was incubated for two hours at 37°C with buffered normal pool plasma. Higher inhibitor titres were measured in 5 and 15BU/mL samples when assays were performed with the Nijmegen method and FVIII-DP without VWF. When samples were diluted in imidazole buffer, similar inhibitor titres, close to expected values, were measured whether VWF was present in the FVIII-DP or not. The data obtained were also more accurate when residual FVIII activity levels between 40% and 60% were used to calculate inhibitor titres, despite a linear type I reaction kinetics. These results support the hypothesis that reliable FVIII inhibitor titres can be measured without the use of FVIII-DP containing VWF when an imidazole-modified assay is used.

Water-repellent-treated enzymatic electrode for passive air-breathing biocathodic reduction of oxygen

A passive-type air-breathing biocathode using bilirubin oxidase as enzyme, ferricyanide as redox mediator, and poly-l-lysine as linker was investigated. Water-repellents from Scotchgard™ with dissolved in methyl isobutyl ketone were directly applied to the enzyme- and mediator-modified biocathode. The partially water-repelled biocathode was placed on a gas-permeable polytetrafluoroethylene (PTFE) membrane and immersed in a buffer solution, and O2 was passively supplied to the biocathode through the PTFE membrane from air. Cathodic currents were dependent on the buffer concentration and amount of applied water repellent. Steady-state catalytic current densities reached 24 and 32mAcm−2 at 0V vs. Ag|AgCl under air and oxygen atmospheric conditions, respectively, using a biocathode modified with 0.32mgcm−2 of the water repellent and 2.0M imidazole buffer at pH7.

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2*

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca(2+)-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.

Transgenic Mouse α- and β-Cardiac Myosins Containing the R403Q Mutation Show Isoform-dependent Transient Kinetic Differences

Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the β-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express α-MHC, whereas the ventricles of larger mammals express predominantly β-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous α-MHC was partially replaced with transgenically encoded β-MHC or α-MHC. A His6 tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for α-S1 (100 μM) is ~ 4-fold weaker than for β-S1 (25 μM). Correspondingly, the MgADP release rate for α-S1 (350 s(-1)) is ~3-fold greater than for β-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for β-S1, but R403Q in α-S1 caused the ADP release rate to increase by 20% (430 s(-1)). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.

Casein maps: Effect of ethanol, pH, temperature, and CaCl2 on the particle size of reconstituted casein micelles

Although conditions favoring casein micelle aggregation are well known, factors promoting the dissociation of the casein micelle are not fully understood. It was our objective to investigate the ethanol-induced dissociation of micellar casein as affected by temperature and a wide range of pH, along with the concentrations of calcium and casein. Two different concentrations of casein micelles were dispersed in imidazole buffer with 0 to 80% ethanol (vol/vol) and 2 and 10mM calcium. Apparent micelle size was determined by dynamic light scattering at 5, 30, and 60°C. In the absence of ethanol, casein precipitation occurred at pH 4.6 in imidazole buffer. Ten to forty percent ethanol promoted casein aggregation (>1,000nm) and higher temperature (30 and 60°C) enhanced this effect. Higher ethanol concentrations at 50 to 80% induced the dissociation (<40nm) of the casein micelle upon acidification (pH <5) and alkalization (pH>8) in imidazole buffer. In addition, higher concentrations of casein (0.25mg/mL) and calcium (20mM) caused the formation of larger aggregates (>1,000nm) in the presence of ethanol when comparing with the initial lower concentrations of casein (0.1mg/mL) and calcium (2mM). Casein micelle dissociation can be achieved near the isoelectric pH by modifying the solvent composition and temperature.

Plasma apolipoprotein O level increased in the patients with acute coronary syndrome

Apolipoprotein (apo) O is a novel apolipoprotein that is present predominantly in high density lipoprotein (HDL). However, overexpression of apoO does not impact on plasma HDL levels or functionality in human apoA-I transgenic mice. Thus, the physiological function of apoO is not yet known. In the present study, we investigated relationships between plasma apoO levels and high-sensitive C-reactive protein (hs-CRP) levels, as well as other lipid parameters in healthy subjects (n = 111) and patients with established acute coronary syndrome (ACS) (n = 50). ApoO was measured by the sandwich dot-blot technique with recombinant apoO as a protein standard. Mean apoO level in healthy subjects was 2.21 ± 0.83 µg/ml whereas it was 4.94 ± 1.59 µg/ml in ACS patients. There were significant differences in plasma level of apoO between two groups (P < 0.001). In univariate analysis, apoO correlated significantly with lg(hsCRP) (r = 0.48, P < 0.001) in ACS patients. Notably, no significant correlation between apoO and other lipid parameters was observed. Logistic regression analysis showed that plasma apoO level was an independent predictor of ACS (OR = 5.61, 95% CI 2.16–14.60, P < 0.001). In conclusion, apoO increased in ACS patients, and may be regarded as an independent inflammatory predictor of ACS patients.