Study on the secondary structure and hydration effect of human serum albumin under acidic pH and ethanol perturbation with IR/NIR spectroscopy

Human serum albumin(HSA)is the most abundant protein in plasma and plays an essential physiological role in the human body.Ethanol precipitation is the most widely used way to obtain HSA,and pH and ethanol are crucial factors affecting the process.In this study,infrared(IR)spectroscopy and near-infrared(NIR)spectroscopy in combination with chemometrics were used to investigate the changes in the secondary structure and hydration of HSA at acidic pH(5.6-3.2)and isoelectric pH when ethanol concentration was varied from 0%to 40%as a perturbation.IR spectroscopy combined with the two-dimensional correlation spectroscopy(2DCOS)analysis for acid pH system proved that the secondary structure of HSA changed significantly when pH was around 4.5.What's more,the IR spectroscopy and 2DCOS analysis showed different secondary structure forms under different ethanol concentrations at the isoelectric pH.For the hydration effect analysis,NIR spectroscopy combined with the McCabe-Fisher method and aquaphotomics showed that the free hydrogen-bonded water fluctuates dynamically,with ethanol at 0-20%enhancing the hydrogen-bonded water clusters,while weak hydrogen-bonded water clusters were formed when the ethanol concentration increased continuously from 20%to 30%.These measurements provide new insights into the structural changes and changes in the hydration behavior of HSA,revealing the dynamic process of protein purification,and providing a theoretical basis for the selection of HSA alcoholic precipitation process parameters,as well as for further studies of complex biological systems.

Combined fluorescence and electrochemical investigation on the binding interaction between organic acid and human serum albumin

Human serum albumin （HSA） is a plasma protein responsible for the binding and transport of fatty acids and a variety of exogenous chemicals such as drugs and environmental pollutants. Such binding plays a crucial role in determining the ADME （absorption, distribution, metabolism, and excretion） and bioavailability of the pollutants. The binding interaction between HSA and acetic acid （C2）, octanoic acid （C8） and dodecanoic acid （C12） has been investigated by the combination of site-specific fluorescent probe, tryptophan intrinsic fluorescence and tyrosine electrochemistry. For the study of the fatty acid interaction with the two drug-binding sites on HSA, two fluorescent probes, dansylamide and dansyl-L-proline were employed in the displacement measurements. Intrinsic fluorescence of tryptophan in HSA was monitored upon addition of the fatty acids into HSA. Electrocatalyzed response of the tyrosine residues in HSA by a redox mediator was used to investigate the binding interaction. Qualitatively, observations from these three approaches were very similar. HSA did not show any change in the fluorescence and electrochemical experiments after mixing with C2, suggesting there is no significant interaction with the short-chain fatty acid. For C8, the measured signal dropped in a single-exponential mode, indicating an independent and non-cooperative binding. The calculated association constant and binding ratio were 3.1 × 10^6 L/mol and 1 with drug binding Site Ⅰ, 1.1 × 107 L/mol and 1 with Site Ⅱ, and 7.0× 0^4 L/mol and 4 with the tryptophan site, respectively. The measurements with C12 displayed multiple phases of fluorescence change, suggesting cooperativity and allosteric effect of the C12 binding. These results correlate well with those obtained by the established methods, and validate the new approach as a viable tool to study the interactions of environmental pollutants with biological molecules.

Antioxidant Effects of Oats Avenanthramides on Human Serum

The antioxidant ability of capsules containing oats avenanthramides on human body was evaluated in present study.Healthy people were randomized to supplementation with oats-derived avenanthramides capsules or placebo for 1 mon.Plasma lipid peroxides and antioxidant status were measured.For 8 capsules （containing 3.12 mg avenanthramides） groups,the levels of serum superoxide dismutase （SOD） and reduced glutathione hormone （GSH） were significantly increased by 8.4 and 17.9%,respectively （P0.05）,and malondialdehyde （MDA） level significantly decreased by 28.1%.The total cholesterol （TC）,triglyceride （TG）,and low density lipoprotein cholesterol （LDL-C） levels were lowered by 11.1,28.1,and 15.1%,respectively （P0.05）.The high density blood lipoprotein cholesterol （HDL-C） levels in the same treat was increased by 13.2%.Based on our research,it can be concluded that oats extract containing avenanthramides possessed a high antioxidative activity on humans.It indicated that oat avenanthramides could be used to prevent hyperlipemic and angiocardiopathy.

Determination of Paraquat in Human Serum by HPLC

目的：建立HPLC法测定人血清中百草枯含量。方法：色谱柱：Kromasil C18柱（200mm×4．6mm，5μm），流动相：乙腈-水（含0．03mol·L-1庚烷磺酸钠，0．24mol·L-1磷酸）=3：97（用三乙胺调pH至2．0）；检测波长258nm；柱温25℃；进样体积20μl；流速0．8ml·min-1。结果：百草枯在0．106—10．6mg·L-1浓度范围内线性关系良好（r=0．9993），最低检出浓度为0．065mg·L-1。高、中、低3种浓度样品绝对回收率〉89．4％，方法回收率〉94．4％，日内精密度RSD在0．12％～1．74％之间，日间精密度RSD在0．44％～2．89％之间。结论：该方法操作简便易行、灵敏度高、专属性强，适用于百草枯的人体血清浓度测定。

Transferrin and Its Isoforms from Normal Human Serum Revealed by Several Analytical Techniques

Transferrin（TF） and its isoforms have been widely reported via various analytical techniques, including a noticeable increased number of isoforms with low content of sialic acid（asialo-, monosialo-, and disialo-transferrin） and asialo-TF as well as disialo-TF, with one or several oligosaccharides released in human serum transferrin（hTf）. Here, hTf has been purified by native gradient polyacrylamide gel electrophoresis（PAGEso） before use. The hTf extracted with the electron-transfer approach showed a single subunit band（77.1 Da） in the SDS-PAGE gel, but it exhibited two bands in the native and denatured isoelectric focusing（IEF） gels, namely, hTf-2Fe^3＋ and apo-hTf, without finding any other transferrin isoforms. A reversed phase HPLC（RP-HPLC） equipped with a C18 column effectively separated hTf and its polymers and combined off-line techniques, including peptide mass fingerprinting（PMF）, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF-MS） and database search, and identified the high homology among hTf, apo-hTf, and their isoforms. Moreover, the elution solution consisting of acetonitrile and formic acid could easily denature both hTf and apo-hTf to form various isoforms during separation with HPLC, indicating that chemical factors lead to the formation of various isoforms in transferrin, artificially, during extraction and separation. The authors claimed that only two transferrin isoforms existed in the NHS, namely, hTf-2Fe^3＋ and apo-hTf, which could be employed in biomarkers, to distinguish the healthy population from many disease sufferers, such as, carbohydrate-deficient transferrin（CDT）

Thermodynamic study on the interaction between anti-tumor drug tegafur and human serum albumin

The changes of thermodynamic properties of the system on interaction between tegafur and human serum albumin （HSA） and the changes of secondary structure units of HSA in the system at 298.15 K have been investigated by the Nano-Watt-Scale isothermal titration calorimetry （ITC）, the Langmuirs binding model and the circular dichroism （CD） spectrometry.

Investigation on the differences of four flavonoids with similar structure binding to human serum albumin

Flavonoids are structurally diverse and the most ubiquitous groups of polyphenols distributed in the various plants,which possess intensive biological activities.In this study,the interaction mechanisms between four flavonoids containing one glucose unit with similar molecular weight isolated from the Tibetan medicinal herb Pyrethrum tatsienense,namely.apigenin-7-O-β-D-glucoside（1）,luteolin-7-O-β-D-glucoside（2）.quercetin-7-O-β-D-glucoside（3）.quercetin-3-O-β-D-glycoside（4）.and human serum albumin（HSA）,were investigated by fluorescence.UV-vis absorbance,circular dichroism,and molecular modeling.The effects of biological metal ions Mg2＋,Zn2＋,and Cu2＋ on the binding affinity between flavonoids and HSA were further examined.Structure-activity relationships of four flavonoids binding to HSA were discussed in depth and some meaningful conclusions have been drawn by the experiment data and theoretical simulation.In addition,an interesting phenomenon was observed that the microenvironment of the binding site I in HSA has hardly changed in the presence of 4 differentiating from the other three flavonoids on the basis of conformation investigations.

Characterization of Gallic Acid Interaction with Human Serum Albumin by Spectral and Molecular Modeling Methods

The binding of drugs with human serum albumin（HSA） is a crucial factor influencing the distribution and bioactivity of drugs in the body.To understand the action mechanisms between gallic acid（GA,3,4,5-trihydroxybenzoic acid） and HSA,the binding of GA with HSA was investigated by a combined experimental and computational approach.The fluorescence properties of HSA and the binding parameters of GA collectively indicate that the binding is characterized by static quenching mechanism at one high affinity binding site.According to the estimated molecular distance between the donor（HSA） and the acceptor（GA）,the binding is related to the fluorescence resonance energy transfer.As indicated by the thermodynamic parameters,hydrophobic interaction plays a major role in the GA-HSA complex.Further,the experimental results reveal that GA is bound in the large hydrophobic cavity of subdomain IIA in the site I of HSA,which is well approved by molecular docking.

On-line Sweeping Sample Concentration in Micellar Electrokinetic Chromatography with Enhanced Sensitivity for the Deter-mination of Carbamazepine in Human Serum

A sensitive and simple micellar electrokinetic chromatography （MEKC） method was developed for the determination of antiepileptic drug, carbamazepine （CBZ）, using sweeping on-line concentration method with photodiode array detection. Under the optimal conditions, the calibration curve was linear over a range of 0.5-40μg·mL^-1 for CBZ with a correlation coefficient of 0.998. The detection limit （S/N = 3：1） of CBZ was 0.10 μg·mL^-1. The sweeping-MEKC method has been successfully applied to the analysis of CBZ in human serum.

Assay of Cysteine in Human Serum with Quinine-Ce^(4+) Chemiluminescence System

A sensitive and selective chemiluminescence (CL) method was developed for the determination of cysteine. This method is based on that the weak CL of cysteine oxidized with cerium (IV) can be greatly enhanced by quinine, and the total cysteine in human serum can be detected through simply diluting with water, showing a simpler analytical characteristic.

Electrochemical Determination of Alkaline Phosphatase in Human Serum by Differential Pulse Voltammetry

Differential pulse voltammetry(DPV) was applied to the determination of alkaline phosphatase(ALP) activity in human serum with phenyl phosphate as the substrate. Phenyl phosphate can enzymatically be hydrolyzed to produce phenol which is quantified by DPV at +660 mV(vs.Ag/AgCl) in the concentration range of 2.0_100 μmol/L. The standard curve for ALP is linear over the range from 0.06 to 1000 U/L with a relative standard deviation of 3.0%. The conditions for the enzymatic reaction and voltammetric detection were optimized and the kinetic constants were also examined.The human serum samples were tested by this method and the results were in good agreement with those obtained by the routine p-nitrophenyl phosphate spectrophotometric method.

Non-covalent binding analysis of sulfamethoxazole to human serum albumin:Fluorescence spectroscopy,UV-vis,FT-IR,voltammetric and molecular modeling

This study was designed to examine the interaction of sulfamethoxazole （SMZ） with human serum albumin（HSA）. Spectroscopic analysis of the emission quenching at different temperatures revealed that the quenching mechanism of human serum albumin by SMZ was static mechanism. The binding constant values for the SMZ-HSA system were obtained to be 22,500 L/mol at 288 K, 15,600 L/mol at 298 K, and 8500 L/mol at 308 K. The distance r between donor and acceptor was evaluated according to the theory of Foster energy transfer. The results of spectroscopic analysis and molecular modeling techniques showed that the conformation of human serum albumin had been changed in the presence of SMZ. The thermodynamic parameters, namely enthalpy change （ΔH^0） - 36.0 kJ/mol, entropy change （ΔS^0） - 41.3 Jim01 K and free energy change （ΔG^0） - 23.7 kJ/ mol, were calculated by using van＇t Hoff equation. The effect of common ions on the binding of SMZ to HSA was tested.

Comprehensive overview of human serum albumin glycation in diabetes mellitus

The presence of excess glucose in blood is regarded as a sweet hurt for patients with diabetes.Human serum albumin(HSA)is the most abundant protein in human plasma,which undergoes severe non-enzymatic glycation with glucose in patients with diabetes;this modifies the structure and function of HSA.Furthermore,the advanced glycation end products produced by glycated HSA can cause pathological damage to the human body through various signaling pathways,eventually leading to complications of diabetes.Many potential glycation sites on HSA have different degrees of sensitivity to glucose concentration.This review provides a comprehensive assessment of the in vivo glycation sites of HSA;it also discusses the effects of glycation on the structure and function of HSA.Moreover,it addresses the relationship between HSA glycation and diabetes complications.Finally,it focuses on the value of non-enzymatic glycation of HSA in diabetes-related clinical applications.

Study on a noninvasive method for rapid screening Human Serum albumin injectables by Raman spectroscopy

Human serum albumin(HSA)injectable product is a severely afflicted area on drug safety due to its high price and restricted supply.Raman spectroscopy performances high specificity on HSA detection and it is even possible to determine HSA injectable products noninvasively.In this study,we developed a noninvasive rapid screening method for of HSA injectable products by using portable Raman spectrometer.Qualitative models were established by using principal component analysis combined with classical least squares(PCA-CLS)algorithm,while quanti-tative model was established by using partial least squares(PLS)algorithm.Model transfer in different instruments of both the same and different apparatus modules was further discussed in this paper.A total of 34 HSA injectable samples collected from markets were used for verification.The identification results showed 100%accuracy and the predicted concentrations of those identified as true HSA were consistent with their labeled concentrations.The quantitative results also indicated that model transfer was excellent in the same apparatus modules of Raman spectrometer at all concentration levels,and still good enough in the different apparatus modules although the relative standard deviation(RSD)value showed a little increasing trend at low HSA concentration level.In conclusion,the method was proved to be feasible and efficient for screening HSA injections,especially on its screening speed and the consideration of glass containers.Moreover,with inspiring results on the model transfer,the method could be used as a universal screening mean to different Raman instruments.

Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples

A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.

Investigation of binding behaviour of procainamide hydrochloride with human serum albumin using synchronous,3D fluorescence and circular dichroism

Interaction of procainamide hydrochloride（PAH） with human serum albumin（HSA） is of great significance in understanding the pharmacokinetic and pharmacodynamic mechanisms of the drug. Multi-spectroscopic techniques were used to investigate the binding mode of PAH to HSA and results revealed the presence of static type of quenching mechanism. The number of binding sites, binding constants and thermodynamic parameters were calculated. The results showed a spontaneous binding of PAH to HSA and hydrophobic interactions played a major role. In addition, the distance between PAH and the Trp–214 was estimated employing the F？rster＇s theory. Site marker competitive experiments indicated that the binding of PAH to HSA primarily took place in subdomain IIA（Sudlow＇s site I）. The influence of interference of some common metal ions on the binding of PAH to HSA was studied. Synchronous fluorescence spectra（SFS）, 3D fluorescence spectra and circular dichroism（CD） results indicated the conformational changes in the structure of HSA.

Development of Ultra Fast Liquid Chromatography (UFLC) Method for Fluorescence Detection of Domperidone in Human Serum and Application to Pharmacokinetic Study

A simple and sensitive fluorescence detection of domperidone by ultra fast liquid chromatographic method was developed and validated in human serum. For the evaluation of new drug delivery systems, conducting of pharmacokinetic studies in human volunteers is essential for approval to marketing after preclinical evaluation in animal models. The present method consists of protein precipitation, extraction of analytes from human serum into dichloromethane and separation using reversed-phase C18 column. Propranolol hydrochloride was used as an internal standard and the eluent was monitored by fluorescence detector at excitation 282 and emission 328 nm. The mobile phase used was 62:38 ratio of 10 mM phosphate buffer pH adjusted to 3.1 with OPA and methanol at a flow rate of 1 mL·min-1. The method was evaluated for assay, LLOD, LLOQ, recovery and stability studies. The retention times for domperidone and propranolol hydrochloride were found to be 6.36 and 7.94 minutes respectively. The intraday and inter-day coefficient of variation and percent error values of assay method were less than 5%;mean recovery was more than 96% for each analyte and the method was found to be precise, accurate and specific during study. The method was successfully applied for pharmacokinetic study of immediate and controlled release bioadhesive hot melt extruded buccal patches of domperidone after buccal administration to healthy human volunteers. The Cmax, Tmax, and AUC0–24 of domperidone from immediate and controlled release buccal patches were found to be 129.7 ng·mL-1, 1.5 h, 455.1 ng·h·mL-1 and 145.7 ng·mL-1, 5.25 h, 911.0 ng·h·mL-1 respectively. A simple, sensitive and reliable method for the fluorescence determination of domperidone in human serum by UFLC method was developed and validated.

(E)-2-Benzylidenecycloalkanones XII.*Kinetic Measurement of Bovine and Human Serum Albumine Interaction with Selected Chalcones and Their Cyclic Chalcone Analogues by UV Spectrophotometry

UV-VIS spectroscopic investigations of interaction of bovine and human serum albumin with selected chalcones (1) and their cyclic chalcone analogues: (E)-2-(4’-X-benzylidene-1-tetralones (3), benzosuberones (4) with dimethylamino and methoxy substituents and (E)-2-(2’,4’-dimethox- ybenzylidene)-1-indanone (2) were performed in polar respiration medium. Absorption maxima of the tested compounds were investigated in the presence of bovine and human serum albumin at the 0, 10, 30 and 60 minute timepoints of the interaction. The absorbance of all studied compounds in the presence of proteins decreased after one hour of the reaction. Molecule 4a showed the strongest and fastest kinet initial interaction with both albumins.

Study on the Interaction between Human Serum Albumins and Methyl Pheophorbide-a-Gd

The binding reaction between methyl pheophorbide-a-Gd (MPA-Gd) and Human Serum Albumins (HSA) was studied by fluorescence and UV-Vis absorption spectra. The results indicated that the binding reaction of them was a single static quenching process, MPA-Gd strongly bound HSA, the binding equilibrium constant K0=2.298×105 L·mol-1 at 25 ℃. The shortest binding distance(r) and energy transfer efficiency(E) between donor (HSA) and acceptor (MPA-Gd) was obtained by Frster′s nonradiative energy transfer mechanism as follows: r=4.03 nm, E=0.12. The enthalpy change (ΔH) and entropy change (ΔS) were calculated at 25 and 37 ℃. The results indicated that the hydrogen bonds played major role in the reaction. Furthermore, the displacement experiments indicated that MPA-Gd could bind to the site Ⅱof HSA.

Interaction between a novel intramolecular charge transfer fluorescent probe PFEP and human serum albumin

The interaction mechanism between human serum albumin(HSA) and 1-phenyl-3(fluorenone-2-yl)-5-(9-ethylcarbazole-3-yl)-2- pyrazoline(PFEP) was investigated by fluorescence and absorption titration techniques in combination with molecular modeling method.Stern-Volmer plots at different temperatures proved that PFEP could quench the intrinsic fluorescence of HSA attributed to a static quenching procedure.The association constants were calculated in the range of 1×10~5-8×10~5mol^(-1) at different pH conditions,a...