Mesoscopic gel at low agarose concentration in water: a dynamic light scattering study.

Previous work in our laboratory has shown that at very low agarose concentration in water gelation still occurs within mutually disconnected, high concentration regions generated by spinodal demixing. The freely diffusing particles obtained in these conditions are studied in the present work by depolarized dynamic light scattering and probe diffusion experiments. These particles are found to behave as large (in fact, mesoscopic) polymer fibers entangled in a continuously rearranged mesh with scaling parameters typical of partially flexible, neutral chains. The present results allow specifying the notion of mesoscopic gelation. They also reveal that the same symmetry-breaking mechanism that allows macroscopic gelation at polymer concentrations well below the threshold for random cross-link percolation generates additional and unexpected phenomena.     

Single major polypeptide of a calicivirus: characterization by polyacrylamide gel electrophoresis and stabilization of virions by cross-linking with dimethyl suberimidate.

A calicivirus, San Miguel sea lion virus serotype 4, isolate 15FT, externally labelled with 125I, was shown by gel electrophoresis to possess a single major polypeptide. The polypeptide migrated anomalously upon electrophoresis in two sodium dodecyl sulfate (SDS) systems: more slowly than bovine serum albumin in a continuous phosphate-buffered system and more rapidly than bovine serum albumin in a discontinuous system. Estimated molecular weights in the two systems were approximately 71,000 and 64,000, respectively. There was no clear evidence for a minor virion polypeptide. Treatment of purified San Miguel sea lion virions with dimethyl suberimidate, a cross-linking reagent, preserved virion integrity during long-term storage at 4 degrees C. Oligomeric species of the polypeptide were observed upon electrophoresis of products from cross-linked virions. Based upon a preferred polypeptide molecular weight estimate of 71,000 and distribution of oligomeric species, a calicivirion model with 120 monomeric protein units is proposed as an alternative to a 180-unit model.     

Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation

We investigated the effects of formaldehyde fixation on the secondary structure of isolated proteins (bovine serum albumin, ribonuclease A, and hemoglobin) using high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Whereas thermograms obtained by scanning calorimetry on unfixed purified proteins demonstrated denaturation transitions in the 70-90 degrees C temperature range, the thermograms showed no denaturation transitions in this temperature range when the proteins had been placed in formaldehyde solutions. Thus, fixation destroyed the denaturation transition of bovine serum albumin, ribonuclease A, and hemoglobin. Infrared spectra obtained on the unfixed and fixed proteins were essentially identical. This demonstrates that the "fixed" proteins retain the secondary structure present before fixation. We therefore conclude that the cross-linking of proteins that occurs in the process of formaldehyde fixation "locks in" the secondary structure of these protein molecules.     

Gelation of chemically cross-linked methylcellulose studied by DSC and AFM

Gelation of methylcellulose (MC) and chemically cross-linked MC via urethane linkage (MCPU) with various molecular weights was investigated in a concentration range from 0.1 to 4.0 wt %. On heating of aqueous solution of MC, three transitions, clear sol to turbid sol, sol to gel and phase separation due to water separation were observed. With increasing molecular weight the transition temperatures decrease. In contrast, no effect of molecular weight on the transition temperatures was observed for MCPU. Structural change of water restrained by MC and MCPU was investigated by DSC. Melting and crystallization of water in both series of sample showed no significant difference, however, a molecular weight dependency of the glass transition was observed for MC. The results obtained in this study indicate that hydrophobic aggregation is restricted by cross-linking. Images of atomic force microscopy (AFM) indicated that from 6 to 16 molecules piled in two layers form a bundle. By chemical cross-linking, molecular chains align in the mono layer, molecular bundles consisting of more than 10 molecules coaggregate and form a large flexible ring.     

The Role of Histidine Residues in the Non-Enzyme Covalent Attachment of Glucose and Ascorbic Acid to Protein

Copper ions have been suggested to play a role in the non-covalent glycosylation (glycation) of proteins via transition metal-catalysed oxidations. We have further investigated “autoxidative glycosylation” by comparison of the behaviour of dog and bovine serum albumin with respect to the oxidative reactions of glucose and ascorbate. The proteins possess similar numbers of total amino residues available for glucose attachment but dog serum albumin contains fewer histidine groups and also lacks a high affinity copper-binding site. We find that the higher copper-binding capacity of bovine serum albumin is reflected in a lower rate of ascorbate oxidation as well as less protein oxidative damage than is the case for dog serum albumin. We also observe that modification of bovine serum albumin histidine groups by diethylpyrocarbonate enhances ascorbate-mediated protein fluorophore formation.     

1H- and 2H-NMR study of bovine serum albumin solutions

Frozen, native and denatured bovine serum albumin solutions have been studied with a wide-band NMR pulse spectrometer. Both macromolecular and water protons spin-spin and spin-lattice relaxation times--t2m, t1m, t2w, t1w--have been measured between 170 and 360 K. In the native sample, the t2m process is the tumbling rate of the bovine serum albumin molecules. It gives to the spin-lattice relaxation an omega 0(-2) frequency dependence at room temperature in the studied frequency range, 6-90 MHz. An additional process contributes to t1m-1; it arises from internal backbone or segmental motions and provides a lower frequency behaviour. On denaturation, bovine serum albumin molecules lose their tumbling motion and form a rigid network, while internal backbone motions seem unaffected. Calorimetric Cp measurement confirms the occurrence of a phase transition upon denaturation. 1H and 2H spin-lattice relaxation times of water protons depend mainly on bound water mobility. 1H and 2H t2w depend also on the tertiary structure of bovine serum albumin and on its mobility, because of a fast exchange process between water and some protein protons (or deutons), while a cross-relaxation process between protein and water protons contributes to 1H t1w. Denaturation has no influence on bound water motional properties and bound water population.     

Thermodynamics of protein cross-links

The thermal transitions of native lysozyme and a well-characterized cross-linked derivative of lysozyme [Imoto, T., and Rupley, J. A. (1973), J. Mol. Biol. 80, 657] have been studied in 1.94 M guanidine hydrochloride at pH 2. The observed increase in the melting temperature from 32.4 degrees C for native lysozyme to 61.8 degrees C for the cross-linked derivative corresponds to a calculated 5.2 kcal/mol increase in the free energy of denaturation. This free-energy change is attributed to the decreased entropy of the unfolded polypeptide chain following introduction of a cross-link and is shown to compare well with theoretical predictions. The possibility that an introduction of a cross-link could also affect the enthalpy of an unfolded protein was investigated. The heats of reduction of bovine serum albumin and lysozyme by dithioerythritol in 6 M guanidine hydrochloride were determined and compared to that for the model peptide, oxidized glutathione. The near identity of the observed heats was taken as evidence that the introduction of cross-links into a random-coil protein does not, in general, introduce strain.     

Structure and formation of a stable histidine-based trifunctional cross-link in skin collagen

A stable nonreducible trifunctional cross-linking amino acid has been isolated from mature bovine skin collagen fibrils. Previous cross-link peptide isolations and amino acid analyses indicate the compound has properties identical with those of hydroxyaldolhistidine. Its newly proposed structure was verified using fast atom bombardment mass spectrometry, and 1H and 13C nuclear magnetic resonance. The data indicated that the cross-link consists of the prosthetic groups from one residue each of histidine, hydroxylysine, and lysine. The 1H and 13C nuclear magnetic resonance data indicated that imidazole C-2 of histidine is linked to C-6 of norleucine (epsilon-deaminated lysine residue) which in turn is linked to the C-6 amino group of hydroxylysine. Based on the trivial names for other cross-linking residues found in collagen and elastin it was given the name histidinohydroxylysinonorleucine. In vitro incubation studies for up to 24 weeks, in aqueous solution at physiological pH and ionic strength, using 6-month-old bovine embryo skin demonstrated a one-to-one stoichiometric relationship between the disappearance of the labile reducible bifunctional cross-link dehydrohydroxylysinonorleucine and the appearance of histidinohydroxylsinonorleucine. These results can partially explain the previously observed disappearance of dehydrohydroxylysinonorleucine with chronological age.     

A generalized Flory-Stockmayer kinetic theory of connectivity percolation and rigidity percolation of cytoskeletal networks

Actin networks are essential for living cells to move, reproduce, and sense their environments. The dynamic and rheological behavior of actin networks is modulated by actin-binding proteins such as α-actinin, Arp2/3, and myosin. There is experimental evidence that actin-binding proteins modulate the cooperation of myosin motors by connecting the actin network. In this work, we present an analytical mean field model, using the Flory-Stockmayer theory of gelation, to understand how different actin-binding proteins change the connectivity of the actin filaments as the networks are formed. We follow the kinetics of the networks and estimate the concentrations of actin-binding proteins that are needed to reach connectivity percolation as well as to reach rigidity percolation. We find that Arp2/3 increases the actomyosin connectivity in the network in a non-monotonic way. We also describe how changing the connectivity of actomyosin networks modulates the ability of motors to exert forces, leading to three possible phases of the networks with distinctive dynamical characteristics: a sol phase, a gel phase, and an active phase. Thus, changes in the concentration and activity of actin-binding proteins in cells lead to a phase transition of the actin network, allowing the cells to perform active contraction and change their rheological properties.     

Investigation of Bovine Serum Albumin (BSA) Attachment onto Self-Assembled Monolayers (SAMs) Using Combinatorial Quartz Crystal Microbalance with Dissipation (QCM-D) and Spectroscopic Ellipsometry (SE)

Understanding protein adsorption kinetics to surfaces is of importance for various environmental and biomedical applications. Adsorption of bovine serum albumin to various self-assembled monolayer surfaces including neutral and charged hydrophilic and hydrophobic surfaces was investigated using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. Adsorption of bovine serum albumin varied as a function of surface properties, bovine serum albumin concentration and pH value. Charged surfaces exhibited a greater quantity of bovine serum albumin adsorption, a larger bovine serum albumin layer thickness, and increased density of bovine serum albumin protein compared to neutral surfaces at neutral pH value. The quantity of adsorbed bovine serum albumin protein increased with increasing bovine serum albumin concentration. After equilibrium sorption was reached at pH 7.0, desorption of bovine serum albumin occurred when pH was lowered to 2.0, which is below the isoelectric point of bovine serum albumin. Our data provide further evidence that combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry is a sensitive analytical tool to evaluate attachment and detachment of adsorbed proteins in systems with environmental implications.     

Effects of COR6.6 and COR15am polypeptides encoded by COR (cold-regulated) genes of Arabidopsis thaliana on dehydration-induced phase transitions of phospholipid membranes.

Cold acclimation of Arabidopsis thaliana includes the expression of cold-regulated (COR) genes and the accumulation of COR polypeptides. The hydration characteristics of two COR polypeptides, COR6.6 and COR15am, have been determined and their effects on the dehydration-induced liquid crystalline-to-gel and lamellar-to-hexagonal II phase transitions in phospholipid mixtures have been examined. After dehydration at osmotic pressures between 8 and 150 MPa, the water content of the COR polypeptides was less than that of bovine serum albumin, with COr15am the least hydrated: bovine serum albumin > COR6.6 > COR15am. Neither COR6.6 nor COR15am altered the dehydration-induced gel lamellar --> fluid lamellar phase transition temperature of either dipalmitoylphosphatidylcholine or dioleoylphosphatidylcholine (DOPC). In multilamellar vesicles of dioleoylphosphatidylethanolamine:DOPC (1:1, mol:mol) prepared by either freeze-thaw or reverse-phase evaporation methods, neither COR6.6, COR15am, nor bovine serum albumin altered the incidence of the dehydration-induced formation of the inverted hexagonal phase as a function of osmotic pressure. However, a specific ultrastructural alteration--the formation of a striated surface morphology in the lamellar domains--was observed in mixtures of dioleoylphosphatidylethanolamine:DOPC that were dehydrated in the presence of COR15am. Nevertheless, neither COR6.6 nor COR15am appears to participate in a specific protein-phospholipid interaction that alters the dehydration-induced phase behavior of phospholipid vesicles.     

Study of the serum albumin-polyethyleneglycol interaction to predict the protein partitioning in aqueous two-phase systems

The theoretical framework based only on the excluded volume forces is not enough to explain the bovine serum albumin partitioning behaviour in aqueous biphasic systems. The goal of this work is to look at the phase separation via the polymer effect on the water structure. Our findings suggest that polyethyleneglycol 600-protein interaction is conducted by van der Waals forces between the hydrophobic surfaces from PEG and protein molecules, which implies the rupture of hydrogen bonds from the structured water in their neighbours. Therefore, the protein will concentrate in the most water-structured phase (polyethyleneglycol) in order to reach the minimal free energy condition. When polyethyleneglycol molecular weight increases, its exclusion from protein surface prevails, thus pushing the bovine serum albumin to the bottom phase.     

Influence of preadsorbed milk proteins on adhesion of Listeria monocytogenes to hydrophobic and hydrophilic silica surfaces.

The adsorption of beta-lactoglobulin, bovine serum albumin, alpha-lactalbumin, and beta-casein for 8 h and beta-lactoglobulin and bovine serum albumin for 1 h at silanized silica surfaces of low and high hydrophobicity, followed by incubation in buffer and contact with Listeria monocytogenes, resulted in different numbers of cells adhered per unit of surface area. Adhesion to both surfaces was greatest when beta-lactoglobulin was present and was lowest when bovine serum albumin was present. Preadsorption of alpha-lactalbumin and beta-casein showed an intermediate effect on cell adhesion. Adsorption of beta-lactoglobulin for 1 h resulted in a generally lower number of cells adhered compared with the 8-h adsorption time, while the opposite result was observed with respect to bovine serum albumin. The adhesion data were explainable in terms of the relative rates of arrival to the surface and postadsorptive conformational change among the proteins, in addition to the extent of surface coverage in each case.     

An X-ray diffraction study of alterations in bovine lung surfactant bilayer structures induced by albumin

Lung surfactant (LS) is an extra-cellular lipid-protein system responsible for maintaining low surface tension in the lung and alveolar stability. Serum proteins cause dysfunction of this material, e.g. in adult respiratory distress syndrome (ARDS). BLES is a clinically used LS consisting of most of the lipids and associated proteins from bovine lung lavage. Aqueous phases of BLES at 30% and 70% hydration, with and without 5% by weight of bovine serum albumin (BSA), calculated on the amount of lipids, were studied using X-ray diffraction during cooling from 42 to 5 degrees C. The diffraction curves are consistent with a transition from a lamellar liquid crystalline phase to a gel phase transition at cooling in the interval 30-20 degrees C. The long-spacings correspond to a reduction of the bilayer thickness during this transition. The wide-angle region shows a peak at 4.1 A below 25 degrees C, which is characteristic of the hexagonal chain packing of the gel phase. The perturbation of the bilayers by the presence of BSA seems to induce a significant decrease of the bilayer thickness. Calculations on the observed limits of swelling (taking place in the range 50-60%) indicate that BSA is closely associated with the BLES bilayers, probably due to electrostatic interaction with the cationic surfactant proteins SP-B and SP-C. This study show that the LS lipid structural organizations are extremely susceptible to small amounts of serum albumin, which may have implications in surfactant related lung disease and clinical applications of surfactant therapy.     

Probing perturbation of bovine lung surfactant extracts by albumin using DSC and 2H-NMR

Lung surfactant (LS), a lipid-protein mixture, forms films at the lung air-water interface and prevents alveolar collapse at end expiration. In lung disease and injury, the surface activity of LS is inhibited by leakage of serum proteins such as albumin into the alveolar hypophase. Multilamellar vesicular dispersions of a clinically used replacement, bovine lipid extract surfactant (BLES), to which (2% by weight) chain-perdeuterated dipalmitoylphosphatidycholine (DPPG mixtures-d(62)) had been added, were studied using deuterium-NMR spectroscopy ((2)H-NMR) and differential scanning calorimetry (DSC). DSC scans of BLES showed a broad gel to liquid-crystalline phase transition between 10-35 degrees C, with a temperature of maximum heat flow (T(max)) around 27 degrees C. Incorporation of the DPPC-d(62) into BLES-reconstituted vesicles did not alter the T(max) or the transition range as observed by DSC or the hydrocarbon stretching modes of the lipids observed using infrared spectroscopy. Transition enthalpy change and (2)H-NMR order parameter profiles were not significantly altered by addition of calcium and cholesterol to BLES. (2)H-NMR spectra of the DPPC-d(62) probes in these samples were characteristic of a single average lipid environment at all temperatures. This suggested either continuous ordering of the bilayer through the transition during cooling or averaging of the DPPC-d(62) environment by rapid diffusion between small domains on a short timescale relative to that characteristic of the (2)H-NMR experiment. Addition of 10% by weight of soluble bovine serum albumin (1:0.1, BLES/albumin, dry wt/wt) broadened the transition slightly and resulted in the superposition of (2)H-NMR spectral features characteristic of coexisting fluid and ordered phases. This suggests the persistence of phase-separated domains throughout the transition regime (5-35 degrees C) of BLES with albumin. The study suggests albumin can cause segregation of protein bound-lipid domains in surfactant at NMR timescales (10(-5) s). Persistent phase separation at physiological temperature may provide for a basis for loss of surface activity of surfactant in dysfunction and disease.     

The antibody–antigen interaction at an aqueous–solid interface: A study by means of polarized infrared ATR spectroscopy

The determination of structural changes in antibodies due to their specific interaction with antigenic proteins is an important problem in understanding immunological responses. The method of polarized ATR infrared spectroscopy applied to protein films adsorbed on an appropriate solid surface can give information about the conformation of the polypeptide chains, as well as their orientation with respect to the surface. The adsorption of anti-rabbit serum albumin onto monomolecular films of rabbit serum albumin, bovine serum albumin, and ovalbumin, and of anti-ovalbumin onto films of rabbit serum albumin and ovalbumin at a Ge-aqueous interface have been studied by this technique. The intensity of the amide I absorption indicates that the strengths of binding of these three albumin proteins with anti-rabbit serum albumin is, under appropriate conditions, in the order rabbit > bovine ? ovalbumin; with anti-ovalbumin, it is ovalbumin ? rabbit. Since the frequencies of the amide I band appear near 1655 cm^?1 for all the proteins and protein complexes studied, the major contributions to their conformation comes from α-helix and random-coil structures. The average orientation of the transition moments of the amide I and A bands has been shown to be about 75° with respect to the surface normal. This indicates that the polypeptides chains are on the average approximately parallel to the surface for all the systems studied. Consequently, the effect of the specific antibody-antigen interaction on the conformation and orientation of the former seems negligible in these films.     

o-Quinones formed in plant extracts. Their reaction with bovine serum albumin

1. The reactions between chlorogenoquinone, the o-quinone formed during the oxidation of chlorogenic acid, and bovine serum albumin depend on the ratio of reactants. 2. When the serum albumin is in excess, oxygen is not absorbed and the products are colourless. This reaction probably involves the thiol group of bovine serum albumin; it does not occur with bovine serum albumin which has been treated with p-chloromercuribenzoate, iodoacetamide or Ellman's reagent. 3. When bovine serum albumin reacts with excess of chlorogenoquinone, oxygen is absorbed and the products are red. The red colour is probably formed by reaction of the lysine in-amino groups of bovine serum albumin, as it is prevented by treating the protein with formaldehyde, succinic anhydride or O-methylisourea. 4. Bovine serum albumin modified by a 1.5-fold (BSA-Q) and a fivefold (BSA-Q2) excess of chlorogenoquinone were separated by chromatography on DEAE-Sephadex A-50, and some of their properties observed. 5. Reaction of BSA-Q2 with fluorodinitrobenzene suggests that the terminal alpha-amino group, as well as lysine in-amino groups, are combined with chlorogenoquinone.     

Quenching of the emission of tryptophan, tyrosine, and serum albumins by cupric ion

The effect of cupric ion on the emission of tryptophan, tyrosine, and serum albumins is studied by emission spectroscopy and lifetime measurements. It is found that whenever cupric ion is bound to tryptophan or tyrosine, their emissions are quenched completely. The quenching may be due to an electron transfer mechanism. The fluorescence of complexes of cupric ions with serum albumins is partially quenched; this is because energy is transferred from tryptophan to the complexed cupric ions by a dipolar energy transfer mechanism. It is deduced from the present study that the tryptophan in the human serum albumin molecule is between 11 and 16 Å from the nearest eupric ion binding sites (assumed to be at the surface of the protein) and that one of the tryptophan in the bovine serum albumin molecule is very close to the cupric ion binding sites and the other is near the center of the bovine serum albumin molecule. It is also found that the deuterium solvent effect on serum albumin fluorescence is very small, and that the quenching of bovine serum albumin fluorescence at the N-F transition is the result of quenching of the fluorescence of both tryptophans. The phosphorescence lifetime apparatus, capable of measuring decay times of signals with intensities changing over a few orders of magnitude, and the ratio spectrofluorometer, both of which were constructed in this laboratory, are also described.     

The binding of flavopiridol to blood serum albumin

Flavopiridol is a potent cyclin-dependant kinase (CDK) inhibitor and is in clinical trials for anticancer treatment. A limiting factor in its drug development has been the high dosage required in human clinical trials. The high dosage is suggested to be necessary because of significant flavopiridol binding to human blood serum. Albumin is the major protein component of blood serum and has been suggested as a likely high affinity binding target. We characterized the binding of human serum albumin to flavopiridol using circular dichroism (hereafter CD). Flavopiridol bound to human serum albumin has a diagnostic CD binding peak at 284 nm. The diagnostic CD binding peak was unobservable for flavopiridol with bovine serum albumin, using the same experimental conditions. However, under higher albumin concentrations a small CD signal is observed confirming, flavopiridol binds to bovine serum albumin as well.     

Bilirubin diffusion through lipid membranes

The possibility that bilirubin can diffuse through lipid bilayers is investigated with liposomes prepared from dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (egg PC) with 22 mole percent cholesterol, and a lipid extract preparation from N115 neuroblastoma cells. Liposomes were prepared with internalized bilirubin and bovine or human serum albumin, and bilirubin efflux into an exogenous solution of human serum albumin was measured. Efflux from DPPC liposomes was significantly higher above the phase transition temperature than below it. This change was dependent on the lipid undergoing a phase transition and could not be accounted for by 6 K change in temperature. Maximum bilirubin efflux from egg PC-cholesterol liposomes was found to depend on the relative internal and external albumin pools, suggesting an equilibrium distribution of bilirubin between them. These observations demonstrate that bilirubin can diffuse freely through these lipid membranes.