On the hydrodynamics and temperature dependence of the solution conformation of human serum albumin from viscometry approach

The paper presents the results of viscosity determinations on aqueous solutions of human serum albumin (HSA) at a wide range of concentrations and at temperatures ranging from 5 to 45 degrees C. On the basis of a modified Arrhenius formula and Mooney's equation, the viscosity-temperature and viscosity-concentration dependence of the solutions are discussed. The effective specific volume, the activation energy and entropy of viscous flow for hydrated HSA were calculated. Different models of HSA molecule are discussed and the best one-from the hydrodynamic point of view-was established. At low concentration limit, such rheological quantities as the intrinsic viscosity and Huggins coefficient were obtained. Using the dimensionless parameter [eta]c, the existence of three characteristic ranges of concentrations: diluted, semi-diluted and concentrated, was shown.     

Viscosity analysis of the temperature dependence of the solution conformation of ovalbumin

The viscosity of ovalbumin aqueous solutions was studied as a function of temperature and of protein concentration. Viscosity-temperature dependence was discussed on the basis of the modified Arrhenius formula at temperatures ranging from 5 to 55 degrees C. The activation energy of viscous flow for hydrated and unhydrated ovalbumin was calculated. Viscosity-concentration dependence, in turn, was discussed on the basis of Mooney equation. It has been shown that the shape parameter S decreases with increasing temperature, and self-crowding factor K does not depend on temperature. At low concentration limit the numerical values of the intrinsic viscosity and of Huggins coefficient were calculated. A master curve relating the specific viscosity etasp to the reduced concentration c[eta], over the whole range of temperature, was obtained and the three ranges of concentrations: diluted, semi-diluted and concentrated, are discussed. It has been proved that the Mark-Houvink-Kuhn-Sakurada (MHKS) exponent for ovalbumin does not depend on temperature.     

A comparative study on viscosity of human, bovine and pig IgG immunoglobulins in aqueous solutions.

This paper presents the results of viscosity determinations on aqueous solutions of human, bovine and pig IgG immunoglobulins over a wide range of concentrations and at temperatures ranging from 5 degrees C to 55 degrees C. On the basis of the generalized Arrhenius formula, the viscosity temperature and the viscosity concentration dependence of the solutions are discussed. By applying an asymptotic form of the generalized Arrhenius formula, such rheological quantities as the intrinsic viscosity and Huggins coefficient were calculated.     

Dynamics of initial L-cell adhesion. II. Competition of albumin and serum activating factor

The initial attachment process of L cells is studied by the combined action of serum and albumin. Both the substances are added jointly in the Eagle medium or one of them was adsorbed on the substrate previously. The results show that there are two factors in the serum: one depressing the cell attachment, like albumin, and the other being just opposite. The simple kinetical competition model is suggested to describe the experimental dependence of final level of attachment on the concentrations of serum and albumin. The examination of the thermal resistance of the serum factor is made; the previous heating to 60-100 degrees C increases the depressing effect of serum and albumin.     

Determination of Some Hydrodynamic Parameters of Ovine Serum Albumin Solutions Using Viscometric Measurements

The influence of protein concentration and temperature on the viscosity of ovine serum albumin (OSA) solutions was studied. The Mooney equation and a modified Arrhenius formula were used to described the viscosity-concentration and viscosity-temperature dependence of the solutions, respectively. The effective specific volume, the activation energy and entropy of viscous flow for hydrated OSA were calculated. The axial ratio and the dimensions of the main semi-axes of hydrated OSA were established. At low concentration limit, the temperature dependence of the intrinsic viscosity and Huggins coefficient is presented. Comparison of some hydrodynamic parameters obtained for different proteins has been made.     

Viscosity of concentrated solutions and of human erythrocyte cytoplasm determined from NMR measurement of molecular correlation times. The dependence of viscosity on cell volume

Metabolically active human erythrocytes were incubated with [alpha-13C]glycine which led to the specific enrichment of intracellular glutathione. The cells were then studied using 13C-NMR in which the longitudinal relaxation times (T1) and nuclear Overhauser enhancements of the free glycine and glutathione were measured. The T1 values of labelled glycine were also determined in various-concentration solutions of bovine serum albumin and glycerol and also of the natural abundance 13C of glycerol in glycerol solutions. From the T1 estimates the rotational correlation time (tau r) was calculated using a formula based on a model of an isotropic spherical rotor or that of a symmetrical ellipsoidal rotor; for glycine the differences in estimates of tau r obtained using the two models were not significant. From the correlation times and by use of the Stokes-Einstein equations viscosity and translational diffusion coefficients were calculated; thus comment can be made on the likelihood of diffusion control of certain enzyme-catalysed reactions in the erythrocyte. Bulk viscosities of the erythrocyte cytoplasm and the above-mentioned solutions were measured using Ostwald capillary viscometry. Large differences existed between the latter viscosity estimates and those based upon NMR-T1 measurements. We derived an equation from the theory of the viscosity of concentrated solutions which contains two phenomenological interaction parameters, a 'shape' factor and a 'volume' factor; it was fitted to data relating to the concentration dependence of viscosity measured by both methods. We showed, by using the equation and interaction-parameter estimates for a particular probe molecule in a particular solution, that it was possible to correlate NMR viscosity and bulk viscosity; in other words, given an estimate of the bulk viscosity, it was possible to calculate the NMR 'micro' viscosity or vice versa. However, the values of the interaction parameters depend upon the relative sizes of the probe and solute molecules and must be separately determined for each probe-solute-solvent system. Under various conditions of extracellular osmotic pressure, erythrocytes change volume and thus the viscosity of the intracellular milieu is altered. The volume changes resulted in changes in the T1 of [alpha-13C]glycine. Conversely, we showed that alterations in T1, when appropriately calibrated, could be used for monitoring changes in volume of metabolically active cells.     

Protein--non-ionic detergent interaction. Interaction of bovine serum albumin with alkyl glucosides studied by equilibrium dialysis and infrared spectroscopy.

The binding isotherms of bovine serum albumin with octylglucoside and decyl glucoside were determined at 7 degrees C and 25 degrees C at pH 7.4 and ionic strength 0.1 M. The average number of detergent molecules bound was found to increase with increasing hydrocarbon chain length. Competitive binding indicates that alkylglycosides combine with the same sites as alkyl sulphates. Native bovine serum albumin has about 12 and 10 sites for non-ionic ligands at 7 degrees C and about 15 and 13 sites at 25 degrees C for octyl and decyl glucosides respectively. The values for standard free energy changes--delta G0, were calculated from the intrinsic association constants. Fourier-transformed infrared spectroscopy was used to study the effects of alkyl glucosides on the conformation of albumin. The results obtained indicate that there are no significant changes in protein structure.     

Effect of dextran and dextran modifications on the thermal and proteolytic stability of conjugated bovine testis beta-galactosidase and human serum albumin

In order to study carbohydrate-induced protein stabilization bovine testis beta-galactosidase and human serum albumin were conjugated with dextran, partially acetylated dextran and partially methylated dextran. The conjugates and the free proteins were compared with respect to thermal stability at 50 degrees C and resistance to proteolytic digestion by subtilopeptidase A. Both beta-galactosidase and serum albumin were stabilized by conjugation with polysaccharide. However, higher stability was achieved by conjugating the proteins with the hydrophilic polysaccharides, dextran and acetylated dextran, than by conjugation with the hydrophobic polysaccharide, methylated dextran. The results are discussed in relation to possible explanations of carbohydrate-induced protein stabilization.     

The role of epsilon-amino groups of lysine of human serum albumin in heat-induced protein denaturation. Increased stability of glycosylated and alkylated albumin in aggregation during heating

Human serum albumin was glycosidated by prolonged protein incubation in phosphate buffer, pH 6.8-7.0, with excess glucose at 37 degrees C. epsilon-amino groups of lysine residues of the albumin molecule were alkylated by pyridoxal-5-phosphate in the presence of NaBH4. The solutions of glycosidated and alkylated serum albumin were incubated at different temperature values in the range of 20 to 80 degrees C in phosphate buffer, pH 7.0, over 30 min. The nondenatured monomer and the resulting aggregated were isolated by TSK-HW-55-gel column chromatography and polyacrylamide gel electrophoresis. The stability of modified proteins elevated in parallel to the increase in the number of the ligand molecules covalently bound to albumin amino groups. The 1-3% aqueous solutions of glycosidated serum albumin containing 3-4 glucose residues and those of alkylated albumin containing 6-7 residues of pyridoxal-5-phosphate were stable on heating up to 80 degrees C and did not form aggregates. Under these conditions the initial serum albumin completely aggregated. Preincubation of the aggregated albumin with glucose at 37 degrees C resulted in protein "renaturation" to the monomeric form with a small number of dimers and trimers.     

Enhancement of the viscosity of mucin by serum albumin.

The interaction of serum albumin with a model epithelial mucin from pig stomach was explored by rotary viscometry. During 30 min of incubation of human serum albumin(20mg/ml) and pig gastric mucin (8mg/ml) in iso-osmotic buffers at 37 degrees C, the solution became markedly viscous. Viscosity enhancement was proportional to albumin concentration (2-40mg/ml), was most pronounced under conditions of low shear rate (less than 45S-1), and was considerably greater than the additive or multiplicative viscosity values calculated from albumin or mucin solutions measured separately. The viscous mucin-albumin complex was destroyed by high shear rates (greater than 90S-1), but slowly re-formed under zero shear conditions. Elevation of pH (7 to 9), ionic strength (0.1 to 1.0), and addition of disodium EDTA (5mM) did not cause marked or specific alterations in the viscosity of the mixture, suggesting that electrostatic interactions probably do not stabilize mucin-albumin complexes. Urea (7M) and heating (35 to 55 degrees C) caused a major increase in the viscosity of mucin and mucin-albumin mixtures, suggesting that rupture of hydrogen bonds, unfolding and partial denaturation of mucin promotes greater intertangling (possibly hydrophobic interactions) between mucin and albumin molecules. The implications of mucin-albumin interaction in diseases associated with mucus obstruction are briefly discussed.     

Temperature behaviour of viscous flow with proteins

The paper presents the results of viscosity determinations on aqueous solutions of different mammalian serum albumins at a wide range of concentrations and at temperatures ranging from 278 K to 318 K. On the basis of these measurements and a modified Arrhenius equation, the functional dependence of the solution activation energy of viscous flow on temperature was established. The analysis of the results obtained shows that the activation energy decreases with increasing temperature according to a square function for solutions, water molecules, and the albumins studied. The rate at which the activation energy decreases with increasing temperature is different for each albumin and mainly depends on its hydrodynamic radius.     

Stability, pKa and plasma protein binding of roscovitine

In the present investigation, the binding of roscovitine (100, 500 and 1500 ng/mL) to plasma proteins was studied at 25 and 37 degrees C by ultrafiltration and equilibrium dialysis methods. Drug stability in plasma was assessed during a 48 h at 4, 25 and 37 degrees C. The effect of thawing and freezing on drug stability was studied. The pKa of roscovitine was measured using capillary electrophoresis coupled with mass spectrometry. Roscovitine was quantified utilizing liquid chromatography and tandem mass spectrometry. Roscovitine is highly bound to plasma proteins (90%). Binding of roscovitine to human serum albumin was constant (about 90%) within concentration range studied while the binding to alpha1-acid glycoprotein decreased with increasing drug concentration indicating that albumin is more important in clinical settings. However, alpha1-acid glycoprotein might be important when plasma proteins change with disease. Protein binding was higher at 25 degrees C compared to 37 degrees C. The results obtained by equilibrium dialysis were in good agreement with those obtained by ultrafiltration. Roscovitine was stable at all temperatures studied during 48 h. Roscovitine has a pKa of 4.4 showing that the drug mainly acts like a weak mono-base. The results obtained in our studies are important prior to clinical trials and to perform pharmacokinetic studies.     

The properties of bovine serum albumin and chymotrypsinogen A in solvent mixtures containing phenol

1. The optical rotation and reduced viscosity of bovine serum albumin and chymotrypsinogen A in solvents containing phenol, acetic acid and water were studied. 2. The changes brought about in the properties of the proteins by varying the composition of the solvent or by heat treatment in these solvents were established to be reversible. 3. A method for returning the proteins to aqueous media, based on these observations, was worked out. 4. The recovered proteins were shown to be very similar to, if not identical with, the native proteins on the basis of measurements of optical rotation, viscosity, sedimentation, ultraviolet spectroscopy, immunochemical behaviour (serum albumin) and proteolytic activity (chymotrypsinogen A, after activation with trypsin). 5. The importance of the findings for partitioning of polyelectrolytes in the phenol-aqueous buffer systems is discussed.     

Role of lipids in the Neurospora crassa membrane. II. Membrane potential and resistance studies; the effect of altered fatty acid composition on the electrical properties of the cell membrane.

The effect of doubling the saturated fatty acid content on the electrophysiology of Neurospora crassa membranes was studied. Intracellular membrane input resistance (Rm) and potential (Em) were measured for wild-type (w/t) and cel- (Tween 40) organisms as a function of temperature. Over the 0 to 40 degrees C temperature range studied, mean Em values of both w/t and cel- (Tw 40) organisms increased from -160 to -210 mV. This difference is greater than that expected from Nernst potential considerations, indicating an active component of Em. This active component is insensitive to a doubling of the saturated fatty acid content. Rm exhibits a temperature dependence and hysteresis. Averaged data indicate an increase in Rm with decreased temperature. The slope of the temperature dependence varies among individual hyphae. Above 17.5 degrees C cel- (Tw 40) hyphae averaged greater than 70% higher values of Rm than w/t. Below 17.5 degrees C w/t Rm data divided into low and high temperature dependence groups, while cel- data exhibited a low temperature dependence. The results are discussed in relation to gel-liquid crystal phase transitions, membrane fluidity, and the contribution of fatty acid structure to membrane electrical properties.     

The use of the method of anomalous time dependence of viscosity for registration of changes in the nucleoide conformation. Part 1

The sensitivity of the method of anomalous time dependence of viscosity to changes in the conformation of DNA-protein complexes (such as nucleoide) by the action of increased temperature (33, 70 and 85 degrees C) and the combined action of temperature and Na+, Cl- ions on lysates of Escherichia coli AB1157 cells has been studied. The optimal conditions of the cell lysis was determined on the basis of the curve parameters of the anomalous time dependence of viscosity.     

Influence of temperature on the determination of enzyme activities in human serum. gamma-Glutamyl transferase.

Optimum reaction conditions for determination of gamma-glutamyl transferase were studied at 25, 30 and 37 degrees C using a kinetic test and gamma-glutamyl-3-carboxy-4-nitranilide as substrate. There was no dependence on temperature of half saturation constants of gamma-glutamyl-3-carboxy-4-nitranilide. The corresponding constants for glycylglycine were influenced by temperature and the pH. The optimum showed a dependence upon temperature. In Arrhenius' plot, a deviation from straight line can be observed only above 35 degrees C. The influence of temperature on the determination of enzyme activities in human serum are discussed.     

Hydration-dehydration of adsorbed protein films studied by AFM and QCM-D

The hydration-dehydration process of an adsorbed human serum albumin film has been studied using atomic force microscopy (AFM) and a quartz crystal microbalance (QCM). All measurements were performed with identically prepared protein films deposited on highly hydrophilic substrates. Both techniques are shown to be suitable for following in situ the kinetics of protein hydration, and for providing quantitative values of the adsorbed adlayer mass. The results obtained by the two methods have been compared and combined to study changes of physical properties of the films in terms of viscosity, shear, Young's modulus, density and film thickness. These properties were found to be reversible during hydration-dehydration cycles.     

Structure and properties of albumin Tokushima and its proteolytic processing by cathepsin B in vitro

Albumin Tokushima is a Japanese genetic variant of human serum albumin. Two homozygous and 6 heterozygous subjects with this variant were found in a family. Albumin Tokushima was purified from sera of the homozygous subjects. Its amino acid composition and amino-terminal sequence were determined and compared with those of a normal serum albumin. Albumin Tokushima with the amino-terminal sequence of Arg-Gly-Val-Phe-His-Arg-Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys- Asp- Leu-Gly-Glu-Glu-Asn-Phe was found to be the same abnormal proalbumin as proalbumin Lille (Abdo, Y. et al. (1981) FEBS Lett. 131, 286-288). The isoelectric points of albumin Tokushima were pH 4.70 and 4.90 as compared with pH 5.05 and 5.25 of a normal serum albumin. Albumin Tokushima was converted to normal serum albumin by purified cathepsin B in vitro. Albumin Tokushima can bind Ni2+ at 4 degrees C but binds little at 37 degrees C.     

Inhibition of glucose 6-phosphatase by pure and impure C-type phospholipases. Reactivation by phospholipid dispersions and protection by serum albumin.

1. Pure or impure C-type phospholipases hydrolysed rat liver microsomal phosphatides in situ at 5 degrees or 37 degrees C. At 5 degrees C mean hydrolysis of total phospholipids was 90% by Bacillus cereus and 75% by Clostridium perfringens (Clostridium welchii) C-type phospholipases. 2. Four degrees of inhibition of glucose 6-phosphatase (D-glucose 6-phosphate phosphohydrolase; EC 3.1.3.9) resulted. (a) At 37 degrees C inhibition was virtually complete and apparently irreversible. (b) At 5 degrees C phospholipase C inhibited 50-87% of the activity expressed by intact control microsomal fractions. (c) Bovine serum albumin present during delipidation alleviated most of this inhibition: at 5 degrees C phospholipase C plus bovine serum albumin inhibited by 0-35% (mean 18%):simultaneous stimulation by the destruction of its latency seems to offset glucose 6-phosphatase inhibition, sometimes completely. (d) If latency was first destroyed, phospholipase C plus bovine serum albumin inhibited 30-50% of total glucose 6-phosphatase activity at 5 degrees C. Only this inhibition is likely largely to reflect the lower availability of phospholipids, essential for maximal enzyme activity, as it is virtually completely reversed by added phospholipid dispersions. Co-dispersions of phosphatidylserine plus phosphatidylcholine (1:1, w/w) were especially effective but Triton X-100 was unable effectively to restore activity. 3. Considerable glucose 6-phosphatase activity survived 240min of treatment with phospholipase C at 5 degrees C, but in the absence of substrate or at physiological glucose 6-phosphate concentrations the delipidated enzyme was completely inactivated within 10min at 37 degrees C. However, 80mM-glucose 6-phosphate stabilized it and phospholipid dispersions substantially restored thermal stability. 4. It is concluded that glucose 6-phosphatase is at least partly phospholipid-dependent, and complete dependence is not excluded. For reasons discussed it is impossible yet to be certain which phospholipid class(es) the enzyme requires for activity.     

The adsorption of proteins and protein--dodecyl sulphate complexes on N-(3-carboxypropionyl)aminodecyl-Sepharose.

Equilibrium and kinetic aspects of the binding of several proteins to N-(3-carboxypropionyl)aminodecyl-Sepharose, an amphiphilic ampholytic adsorbent, were studied at 22 degrees C, pH 7.0, I 0.10--0.12. In the absence of detergents Scatchard plots are linear for human haemoglobin and soya-bean trypsin inhibitor, but non-linear for bovine serum albumin, which is also adsorbed more tightly than the other two proteins. The introducion of 3.5mM-sodium dodecyl sulphate causes dramatic increases in the amounts and affinities of serum albumin and haemoglobin adsorbed, but has relatively little effect on the trypsin inhibitor. At concentrations of sodium dodecyl sulphate greater than about 10mM there is a fall in the binding of all proteins, owing to competition from the detergent for binding sites on the adsorbent, and a tendency towards more uniform behaviour by different proteins. Kinetic experiments suggest that in the absence of the detergent haemoglobin and serum albumin are adsorbed initially by mainly ionic forces, but that subsequently hydrophobic forces become dominant. Addition of 3.5 mM-sodium dodecyl sulphate causes pronounced changes in the time course of adsorption of haemoglobin and serum albumin, the nature of the changes being different for each protein. The significance of these results is discussed.