Effect of water on the temperatures of human immunoglobulin conformation transitions

A differential thermal analysis of native and denaturated human immunoglobulin (G isotype) and mixtures of the native protein with water over the temperature range of 80–570 K is conducted. Temperatures of the protein conformational transitions and the effect of water on them are investigated. The limit of water solubility in the native protein is determined calorimetrically from the enthalpy of excess water phase melting. A physical state diagram of the immunoglobulin-water system over a wide range of temperatures and component concentrations is built and analyzed.     

4.50—Polarographic investigation of conformational changes of human serum albumin: Part I. Unfolding of human serum albumin by urea

The mechanism of the unfolding of human serum albumin by urea was studied using d.c. polarography. It was found that this reaction is a complex process which cannot be described in terms of a two-state transition model. As well as the Brdi?ka catalytic current we have also studied the reduction current of disulfide groups in native and denatured human serum albumin. The number of cystine residues accessible for electrode reduction in native and denatured protein was calculated. On the basis of these results a scheme for the unfolding of human serum albumin by urea is proposed.     

Site-specific Heterogeneity of Multi-domain Human Serum Albumin and its Origin: A Red Edge Excitation Shift Study†

Conformational heterogeneity is a defining characteristic of a protein and is vital in understanding its function and folding landscape. In the present work, we interrogated the presence of conformational heterogeneity in multi-domain human serum albumin in a domain-specific manner using red edge excitation shift (REES) in its native state and also monitored its variation along the unfolding transition. We also looked into the origin of such conformational heterogeneity by varying the solution viscosity. We observed (1) even in the native state, the heterogeneity and dynamics of the side chain exhibit varied behaviors depending on which domain of the multi-domain human serum albumin (HSA) is being examined. (2) When the protein is in the unfolded state, the extent of REES is rendered unimportant since there is a greater quantity of free water present, in addition to the disruption of the protein's structure. (3) While the rigid protein matrix provides the rigidity of domain-I and domain-III, the rigidity of domain-II is provided by water molecules, which indicates that the role of water molecules in providing the rigidity is significant. Overall, our results provide direct evidence of the rigidity and alternate side chain packing arrangement of protein core that varies domain-wise in multi-domain HSA.     

Thiophilic interaction chromatography of serum albumins

An investigation of the binding of native and recombinant human serum albumin and bovine serum albumin on three thiophilic gels, PyS, 2S, and 3S was performed. In addition to these proteins, we studied serum albumins from several species such as goat, rabbit, guinea pig, rat, hamster, baboon, and pig. Our results reveal that recombinant human serum albumin (rHSA) binds completely to PyS whereas native human serum albumin and bovine serum albumin bind only partially to PyS. The binding affinities of rHSA, human serum albumin and bovine serum albumin to 2S and 3S gels are less than their binding to PyS. Serum albumins from goat, rabbit, guinea pig, rat, hamster, baboon, and pig bind much stronger to 3S gel than human and bovine serum albumins. The binding of pig and hamster serum albumins is stronger than that of rat, goat, baboon, and rabbit.     

Modeling the absorption spectrum of tryptophan in proteins

We have applied time-dependent density functional theory (TDDFT) to study the valence pi-pi* excited states of the tryptophan chromophore in the environment of the proteins barnase and human serum albumin. The chromophore is represented by indole. Due to the approximate nature of TDDFT, in the gas phase the calculated vertical transition energies to the 1L valence states are reordered with respect to experiment. The 1L(a) state responds more than the 1L(b) state to the local environment, described fully at the TDDFT level, and to bulk environment, described by a set of point charges. Nevertheless, the vertical transitions are readily identified. For human serum albumin, our calculations predict distinct spectral characteristics between structures with different tryptophan side chain torsion angles. The computational tractability of TDDFT relative to more accurate ab initio methods allows a large part of the surrounding protein environment (up to 100 atoms) to be explicitly included in the TDDFT calculations.     

Serum albumin in 2D: A Langmuir monolayer approach

Understanding of protein interaction at the molecular level raises certain difficulties which is the reason a model membrane system such as the Langmuir monolayer technique was developed. Ubiquitous proteins such as serum albumin comprise 50% of human blood plasma protein content and are involved in many biological functions. The important nature of this class of protein demands that it be studied in detail while modifying the experimental conditions in two dimensions to observe it in all types of environments. While different from bulk colloidal solution work, the two dimensional approach allows for the observation of the interaction between molecules and subphase at the air–water interface. Compiled in this review are studies which highlight the characterization of this protein using various surroundings and also observing the types of interactions it would have when at the biomembrane interface. Free-energy changes between molecules, packing status of the bulk analyte at the interface as well as phase transitions as the monolayer forms a more organized or aggregated state are just some of the characteristics which are observed through the Langmuir technique. This unique methodology demonstrates the chemical behavior and physical behavior of this protein at the phase boundary throughout the compression of the monolayer.     

Thermodynamic and structural changes associated with the interaction of a dirhamnolipid biosurfactant with bovine serum albumin

The interaction of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa with bovine serum albumin was studied by means of various physical techniques. Binding of the biosurfactant to bovine serum albumin was first characterized by isothermal titration calorimetry, showing that one or two molecules of dirhamnolipid, in the monomer state, bound to one molecule of the protein with high affinity. These results were confirmed by surface tension measurements in the absence and presence of bovine serum albumin. As seen by differential scanning calorimetry, dirhamnolipid shifted the temperature of the thermal unfolding of bovine serum albumin toward higher values, thus increasing the stability of the protein on heating. The impact of dirhamnolipid on the structure of the native protein was low, since most of the secondary structure remained unaffected upon interaction with the biosurfactant, as shown by FTIR spectroscopy. However, 2D correlation infrared spectroscopy indicated that the sequence of temperature-induced structural changes in native bovine serum albumin was modified by the presence of the biosurfactant. The consequences of these results in relation to possible applications of these dirhamnolipid biosurfactants for protein studies are discussed.     

Measurement of protein denaturation in human synovial fluid and its analogs using differential scanning calorimetry

Differential scanning calorimetry (DSC) was used to evaluate the thermal transitions associated with protein constituents of synovial fluid samples from three individuals with osteoarthritis. Analysis of the multi-component DSC curves revealed that major endothermic transitions of synovial fluid occur between 60 and 80 °C and can be resolved into three peaks, likely due to the unfolding of human serum albumin and immunoglobulins, and that the enthalpies of these transitions can be quantified in terms of their relative contribution to the total system enthalpy. DSC was also used to analyze a solution of bovine calf serum, a lubricant used in simulator wear testing of joint replacement implants, and the resulting endothermic transitions occurred in a temperature range relevant to that produced by frictional heat during such wear simulator testing. Results of this study indicate a new application for DSC as a direct method for studying thermal stabilities of both bovine calf serum and synovial fluid. The use of DSC is proposed as a diagnostic tool to detect altered thermal properties or protein concentrations indicative of a diseased or injured state, and as a development tool to test the efficacy of additives in controlling protein denaturation associated with increased wear in joint replacement implants.     

Ultrafast solvation dynamics of human serum albumin: correlations with conformational transitions and site-selected recognition

Human serum albumin, the most abundant protein found in blood plasma, transports a great variety of ligands in the circulatory system and undergoes reversible conformational transitions over a wide range of pH values. We report here our systematic studies of solvation dynamics and local rigidity in these conformations using a single intrinsic tryptophan (W214) residue as a local molecular probe. With femtosecond resolution, we observed a robust bimodal distribution of time scales for all conformational isomers. The initial solvation occurs in several picoseconds, representing the local librational/rotational motions, followed by the dynamics, in the tens to hundreds of picoseconds, which result from the more bonded water in the tryptophan crevice. Under the physiological condition of neutral pH, we measured approximately 100 ps for the decay of the solvation correlation function and observed a large wobbling motion at the binding site that is deeply buried in a crevice, revealing the softness of the binding pocket and the large plasticity of the native structure. At acidic pH, the albumin molecule transforms to an extended conformation with a large charge distribution at the surface, and a similar temporal behavior was observed. However, at the basic pH, the protein opens the crevice and tightens its globular structure, and we observed significantly faster dynamics, 25-45 ps. These changes in the solvation dynamics are correlated with the conformational transitions and related to their structural integrity.     

ROTATIONAL DIFFUSION OF RHODAMINE 6G IN HUMAN BLOOD SERUM

Abstract —The time-dependent rotational relaxation of Rhodamine 6G in fresh and dried human blood serum was investigated using picosecond phasefluorometry. Measurements were also carried out in aqueous solutions of amino acids, glucose, urea, and bovine serum albumin to evaluate our model for the interactions. It is shown that the distribution of Rhodamine 6G between the aqueous and the protein phase of the blood serum strongly depends on protein concentration and temperature.     

Development of a Capillary Electrophoresis Method to Monitor Protein Oxidation and Antioxidant Protection

The development of a new capillary zone electrophoresis (CZE) method for the determination of protein oxidation by free radicals is described. The effects of various experimental conditions, such as type of capillary, pH, buffer concentration, capillary dimensions, temperature, applied voltage, and addition of some organic solvents, were investigated. The sample injection procedure was also optimized. Target proteins investigated here (lysozyme, human serum albumin, and β-lactoglobulin A) were effectively damaged by free radicals generated in the aqueous phase from 2,2′-azobis[2-amidinopropane] (AAPH). The new method allowed global protein fragmentation to be quantified as a function of time by monitoring the decrease in peak height of the native protein. Furthermore, the protective efficacy of antioxidants was assessed by concentration-inhibition curves against protein fragmentation. The method is thus suitable to screen compounds able to protect proteins against oxidative damage.     

Protein-lipid interactions at the air/water interface

Surface pressure measurements and external reflection FTIR spectroscopy have been used to probe protein-lipid interactions at the air/water interface. Spread monomolecular layers of stearic acid and phosphocholine were prepared and held at different compressed phase states prior to the introduction of protein to the buffered subphase. Contrasting interfacial behaviour of the proteins, albumin and lysozyme, was observed and revealed the role of both electrostatic and hydrophobic interactions in protein adsorption. The rate of adsorption of lysozyme to the air/water interface increased dramatically in the presence of stearic acid, due to strong electrostatic interactions between the negatively charged stearic acid head group and lysozyme, whose net charge at pH 7 is positive. Introduction of albumin to the subphase resulted in solubilisation of the stearic acid via the formation of an albumin-stearic acid complex and subsequent adsorption of albumin. This observation held for both human and bovine serum albumin. Protein adsorption to a PC layer held at low surface pressure revealed adsorption rates similar to adsorption to the bare air/water interface and suggested very little interaction between the protein and the lipid. For PC layers in their compressed phase state some adsorption of protein occurred after long adsorption times. Structural changes of both lysozyme and albumin were observed during adsorption, but these were dramatically reduced in the presence of a lipid layer compared to that of adsorption to the pure air/water interface.     

Glycation alter serum albumin binding of valsartan and nateglinide when studied contemporarily

In this research work, an attempt was made to study alteration in glycated serum albumin binding of valsartan and nateglinide using validated HPLC-UV method and ultrafiltration as in vitro protein binding study model. The chromatographic conditions involved stationary phase Kromasil-100 C18 (100?×?4.6?mm, 3.5?µm) with mobile phase of 10?mM phosphate buffer, acetonitrile, isopropyl alcohol in the ratio of 30:65:5 as isocratic mode at a flow rate of 0.8?mL/min; and the eluent was monitored at 218?nm. Protein precipitation technique was used to extract the drugs from human plasma. The calibration curve was found linear in the range from 50 to 5000?ng/mL. Glycation of human serum albumin was achieved at different concentration levels using D-(+)-glucose and glycated human serum albumin (Gly-HSA) were prepared. Valsartan and nateglinide were not affected the plasma protein binding of each other when studied using HSA. The unbound fraction of valsartan and nateglinide was increased to 10–20 times when spiked with Gly-HSA. About 20% increase in unbound fraction of valsartan was observed when spiked with 10?µg/mL of nateglinide. Furthermore, the unbound fraction of nateglinide was increased nearly to 10% more when incubated with Gly-HSA as compare to recombinant human serum albumin.     

Study of cryostructuring of polymer systems. 42. Physicochemical properties and microstructure of wide-porous covalently cross-linked albumin cryogels

Chemically cross-linked wide-porous protein cryogels have been obtained by freezing aqueous bovine serum albumin (BSA) solutions (30–50 g/L) at–15,–20, or–25°C in the presence of water-soluble carbodiimide (CDI) as a coupling agent. It has been shown that the gel-fraction yield and the swelling extent of the polymer phase of the formed spongy matrices depend primarily on the initial concentration of albumin and the amount of CDI added to a system, while the morphometric characteristics of the porous microstructure of the BSA cryogels are mainly determined by the temperature of the cryogenic treatment. The sizes of macropores in the obtained cryogels range from ≈50 to ≈200 μm. A high-sensitivity differential scanning calorimetric study of the conformational state of protein macromolecules incorporated into the spatial network of the polymer phase (gel walls of macropores) of the cryogels has shown that, during the cryotropic gelation, the native structure of albumin globules is subjected to “cold denaturation,” and the partly unfolded conformation of the protein is, simultaneously, fixed by intermolecular covalent cross links.     

Size-exclusion chromatographic behavior of bovine serum albumin in uni-uni valent ions solutions

Summary Molecular interaction between uni-uni valent ions and bovine serum albumin was investigated by size-exclusion chromatography. Elution profiles are first presented for salt and protein solutions as samples with water as the mobile phase; then for water and protein as samples with salt solutions as the mobile phase. The results suggest the existence of a dynamic equilibrium between the salt ions and the protein ions as reactants and the ion-pair (salt-protein) complex as a product.     

Development of a disulfiram-modified human serum albumin-based HPLC column

Summary A human serum albumin (HSA)-based HPLC column has been modified in situ by disulfiram, an alcohol-deterrent drug reported to bind cys₃₄, the only free cysteine in HSA, under physiological conditions. The reversible and covalent binding of disulfiram was found to change the binding properties of the protein, giving rise to a new selector which performed differently from the native albumin-based stationary phase. When low concentrations of disulfiram were used as mobile phase modifier, reversible binding resulted in a cooperative allosteric effect with improved selector performance. Covalent modification resulted in markedly reduced affinity for binding of the drugs to sites I or II, while still maintaining enantioselectivity. This study has enabled the monitoring of interactions of disulfiram with potentially coadministered drugs, and the preparation of a chiral selector with different drug affinity and enantioselectivity.     

Pure Protein Scaffolds from Pickering High Internal Phase Emulsion Template

The formation of hierarchical porous protein scaffolds from oil‐in‐water (o/w) high internal phase emulsions (HIPEs) stabilized by bovine serum albumin (BSA) protein nanoparticles (Pickering HIPE) is reported. The route consists of three principal steps. First, a stable o/w HIPE stabilized by BSA protein nanoparticles is formulated. Next, crosslinking the dispersed protein nanoparticles gives rise to a gel in the continuous water phase to freeze the emulsion's microstructure. Finally, removal of the oil components and water directly leads to a three dimensional, bimodal meso‐macroporous protein scaffold, which is suitable for a wide range of biomedical applications.     

Direct detection of native proteins in biological matrices using extractive electrospray ionization mass spectrometry

The high-throughput and sensitive characterization of native proteins in biological samples is of increasing interest in multiple disciplines. Extractive electrospray ionization (EESI) forms ions of native proteins including lysozyme, α-chymotrypsin, myoglobin, human serum albumin, RNAse A and blood hemoglobin in extremely complex biosamples or PBS buffer solutions by softly depositing charges on the protein molecules. This method produces no significant conformational changes of the proteins in the ion formation process, and features direct detection of trace proteins present in biological matrices. The detection limit of low pmol L(-1) for lysozyme in untreated biological liquids such as human urine and tears was demonstrated using EESI mass spectrometry (MS), showing an attractive MS platform for the direct analysis of native proteins in actual biological samples.     

Biological reference materials for assaying human albumin in urine

Summary To assess the interlaboratory variation in the results of albumin measurements, we prepared albumin solutions in human urine at various concentrations within the normal range. Since some investigators have reported that albumin is unstable in some human urine samples stored at –20°C, we screened urine samples from 21 persons to identify samples that were stable under these conditions and that had low native albumin content. The urine of two donors met these criteria, and they provided urine, which we prefiltered, sterile-filtered, and spiked with commercially available human serum albumin. The albumin was characterized as pure by a Lowry assay of protein content with National Institute of Standards and Technology bovine serum albumin (standard reference material 926) as the standard and by the appearance of one band on agarose gel electrophoresis. To evaluate the necessity for additional stabilization when urine samples are stored at –20°C, a surfactant was included in one set of materials and not included in another. The materials with surfactant have been evaluated for 10.5 months and those without surfactant for 5 months. The preserved materials showed no significant loss of activity during this period. The unpreserved materials remained stable for 2 months, and then the two higher level materials appeared to loose activity. The negative slope of the highest level of unpreserved material was statistically significant (p=0.01) during this period. In our laboratory, the albumin recovered by enzyme immunoassay was 106.7% and 115.9% in two preserved normal-range materials and 102.2% and 106.3% in similar unpreserved materials.     

Synthesis and chromatographic properties of an HPLC chiral stationary phase based upon human serum albumin

Summary A new HPLC stationary phase was synthesized by thein situ covalent immobilization of human serum albumin (HSA). The protein was immobilized on a commerically available diol column which had been activated with 1,1-carbonyldiimidazole. Initial chromatographic studies show that this phase can be used for chiral separations of enantiomeric solutes and that these separations may reflectin vitro binding to the HSA. The effects of mobile phase composition and temperature on the stereochemical resolutions are reported.