Concentrations and partial volumes of proteins

The amount of egg albumin and bovine serum albumin in water has been studied as a function of temperature and time. The temperature selected for heating was 102 °C. The proteins appear to decompose above this temperature. The suitable length of time of drying is 24 h at 102 °C. Four modifications of the method of dry weight have been explored. Glass paper in the weighing bottle increases the area available for evaporation. The densities of solutions of egg albumin and bovine serum albumin have been measured at 30.00 °C as a function of concentration with a Mettler/Paar density meter and the apparent specific volumes calculated. The apparent specific volume of egg albumin is independent of concentration and is 0.7463 ± 0.00016. The apparent specific volume of bovine serum albumin is constant from zero concentration up to about 0.2 g/g of solution and in this concentration range the apparent specific volume is 0.7348 ± 0.0001. Beyond this concentration, in agreement with the results of Bernhardt and Pauly, the apparent specific volume drops sharply with increasing protein concentration.     

Protein hydration: A sucrose probe

The hydration of conalbumin, of myoglobin, of lysozyme, of carbon monoxide hemoglobin, of β-lactoglobin, of bovine serum albumin, of ovomucoid, of ribonuclease, and of egg albumin has been measured with equilibrium dialysis using sucrose as the probe at 30 °C. All proteins were at their isoelectric points except lysozymes and β-lactoglobulin and also samples of egg albumin which had been shifted to a more alkaline pH. Departure from their isoelectric points leads to an increase in the apparent protein hydration. Decreasing the temperature to 11.5 °C produces a slight increase in the hydration of egg albumin. A method is proposed for the calculation of protein hydration. The calculated protein hydration tends to be less than that determined experimentally for five of the proteins. There is satisfactory agreement with four of the proteins.     

An automated differential scanning dilatometer.

A sensitive scanning dilatometer has been constructed which continuously records the apparent partial specific volume of small biological samples (10–100 mg) as a function of temperature. Changes in volume of 0.02 μl can be resolved. Tedious calibrations with their inherent errors are eliminated by employing a differential approach in which a recording electrobalance responds to the difference in buoyancy of identical sample and reference tubes. Agreement with the literature values for the specific volume of n-eicosane and the apparent partial specific volume of KCl was excellent. Apparent partial specific volumes obtained for dipalmitoyl lecithin, egg albumin, and bovine serum albumin agreed well with published values.     

Temperature dependence of solute transport and enzyme activities in hog renal brush border membrane vesicles

The temperature dependence of sodium-dependent and sodium-independent d-glucose and phosphate uptake by renal brush border membrane vesicles has been studied under tracer exchange conditions. For sodium-dependent d-glucose and phosphate uptake, discontinuities in the Arrhenius plot were observed. The apparent activation energy for both processes increased at least 4-fold with decreasing temperature. The most striking change in the slope of the Arrhenius plot occurred between 12 and 15°C. The sodium-independent uptake of d-glucose and phosphate showed a linear Arrhenius plot over the temperature range tested (35–5°C). The behavior of the transport processes was compared to the temperature dependence of typical brush border membrane enzymes. Alkaline phosphatase as intrinsic membrane protein showed a nonlinear Arrhenius plot with a transition temperature at 12.4°C. Aminopeptidase M, an extrinsic membrane protein exhibited a linear Arrhenius plot. These data indicate that the sodium-glucose and sodium-phosphate cotransport systems are intrinsic brush border membrane proteins, and that a change in membrane organization alters the activity of a variety of intrinsic membrane proteins simultaneously.     

Water stress preconditioning and cotton root pressure-flux relationships

Summary A model was developed to describe interactive effects of exposure time and treatment on thermostability of excisedIllicium parviflorum Michx. root cell membranes using electrolyte leakage (L_c) procedures. Roots were moved from 25°C to treatment temperatures between 35°C and 60°C for 30 to 300 min. A sigmoidal response described L_c increases with increasing temperature at selected time exposures and the lethal exposure time decreased exponentially as temperature increased. The lethal temperature (52.0±1.1°C) for a 15 min exposure using this technique was comparable to the critical temperature (52.2±1.2°C) when roots were exposed to gradually increasing temperatures (4°C per h). Total protein content of roots began to decrease as temperatures increased from 35 to 40°C and the temperature corresponding to 50% reduction in total proteins was 49.1±2.2°C.     

Partial specific volume changes of proteins densimetric studies

The partial specific volume of proteins has been investigated as a function of protein concentration, temperature, pH, and a number of denaturing and non-denaturing solvent components. Protein concentration (5–100 mg·ml^?1) is found to have no effect; increasing temperature (4–40°C), as well as addition of electrolytes, sugars, and polyols, leads to an approximately linear increase; upon ligand binding, as well as denaturation by heat, extremes of pH, guanidine. HCl, urea, or sodium dodecyl sulfate, a predominantly non-linear decrease is observed. The study demonstrates the importance of measuring rather than calculating the specific volume of proteins. This holds especially in multicomponent systems, where solvent conditions may cause drastic effects.     

25-Hydroxylation of 1-α-hydroxyvitamin D-3 in rat and human liver

1α-Hydroxyvitamin D-3 25-hydroxylase activity was measured in subcellular fractions of rat and human liver. The formation of 1,25-dihydroxyvitamin D-3 was determined by high pressure liquid chromatography. In rat liver 1α-hydroxyvitamin D-3 25-hydroxylase activities were found in the purified nuclei, the heavy mitochondrial fraction and the microsomal fraction. The enrichment of 25-hydroxylase activity was highest in the heavy mitochondrial fraction. With this fraction a minimum amount (about 0.5 mg) of protein was required before formation of 1,25-dihydroxyvitamin D-3 could be detected. Above this amount the reaction was linear with amount of protein up to at least 2 mg/ml. The reaction was also linear with time up to 60 min. An apparent K_m value of 2·10^?5 M was found. The mitochondrial 25-hydroxylase was stimulated by addition of cytosolic protein or bovine serum albumin. The degree of stimulation was dependent on the amount of mitochondrial protein present in the incubation mixture. Maximal stimulation was seen with 0.2 mg/ml of either protein in the presence of 0.5 mg mitochondrial protein. The stimulating effect remained after heating the protein for 5 min at 100°C. The cytosolic protein did not stimulate a reconstituted mitochondrial 1α-hydroxyvitamin D-3 25-hydroxylase. The mitochondrial vitamin D-3 25-hydroxylase was inhibited both by cytosolic protein and by bovine serum albumin. Human liver revealed only one 1α-hydroxyvitamin D-3 25-hydroxylase activity located to the heavy mitochondrial fraction. The results are in agreement with previous studies on the localization of vitamin D-3 25-hydroxylase in rat and human liver. The difference in localization of the 25-hydroxylase between rat and human liver implies that studies on the regulation of the microsomal 25-hydroxylase in rat liver may not be relevant to the situation in human liver.     

The temperature dependence of the redox potential of horse heart cytochrome c in sodium chloride solutions

The E⁰′ values for the conversion of horse heart cytochrome $\text{c}$ from the oxidized to the reduced form as a function of temperature have been measured in 0.10 M NaCl, 0.10 M sodium phosphate, pH 7.0 solutions in H₂O and D₂O. In H₂O, the decrease in the E⁰′ value is linear with increasing temperature up to 42°C. Above this temperature, the decrease is again linear but with a much greater slope. In D₂O solutions, however, this biphasic behavior was not observed but instead a single line was obtained over the temperature range studied (25°C to 50°C). These results are interpreted in terms of the ability of NaCl to cause a destructuring of the bulk H₂O above 42°C but not in the more stable D₂O (Kreishman, Foss, Inoue and Leifer (1976) $\text{Biochemistry}\text{,}\text{15}$, 5431–5435). This decrease in water structure results in a shift in the equilibrium to the larger oxidized form as indicated by the decrease in E⁰′.     

Thermal transitions of proteins

A new method for monitoring the thermal transitions of proteins is described. An unbuffered solution of native protein shows a significant and fairly abrupt change in pH as the protein becomes heat denatured. Suitable plots permit the “melting point” of the protein to be assigned. Twenty proteins have been studied with emphasis on egg albumin. The transition temperature of egg albumin is independent of protein concentration, of pH in the neutral zone, is moderately dependent on the rate of heating, increases with increasing NaCl concentration, varies inversely with the guanidine hydrochloride concentration. There is more than a 35 °C spread in the melting temperatures of the various proteins and no apparent relation exists between the melting temperature of a protein and structural features of the protein.     

Chloroplast thylakoid protein changes induced by low growth temperature in maize revealed by immunocytology

Tissue-specific effects of low growth temperature on maize chloroplast thylakoid protein accumulation were analysed using immunocytology. Sections of leaves from plants grown at 25 and 14°C were probed with antibodies to specific chloroplast thylakoid proteins from the four major protein multisubunit complexes of the thylakoid membrane followed by fluorescein-conjugated goat anti-rabbit antibodies. At a normal growth temperature of 25°C, the 32 kDa D1 protein of the photosystem II reaction centre and the 33 kDa protein of the extrinsic oxygen-evolving complex of photosystem II are both accumulated to a greater degree in the mesophyll than in the bundle sheath chloroplasts. In contrast, subunit II of photosystem I, cytochrome f and the α- and β-subunits of ATP synthetase are predominant in the bundle sheath thylakoids at 25°C. A striking difference between the 25°C-grown and the 14°C-grown leaf tissue was the presence in the latter of (20–30%) cells whose chloroplasts apparently completely lack several of the thylakoid proteins. In plants grown at 14°C, the accumulation of the 33 kDa protein of the extrinsic oxygen-evolving complex of photosystem II was apparently unchanged, but other thylakoid proteins showed a significant reduction. The uneven distribution of proteins between the bundle sheath and mesophyll chloroplasts observed at 25°C was also maintained at 14°C. Reduction in the fluorescence at 14°C was manifested either as an overall reduction in the diffuse fluorescence across the chloroplast profiles or less frequently as a reduction to small discrete bodies of intense fluorescence. The significance of these results to low-temperature-induced reduction in the photosynthetic productivity of maize is discussed.     

Photophysical Probes of the Amorphous Solid State of Proteins

The properties of amorphous solid proteins influence the texture and stability of low-moisture foods, the shelf-life of pharmaceuticals, and the viability of seeds and spores. We have investigated the relationship between molecular mobility and oxygen permeability in dry food protein films—bovine α-lactalbumin (α-La), bovine β-lactoblobulin (β-Lg), bovine serum albumin (BSA), soy 11S globulin, and porcine gelatin—using phosphorescence from the triplet probe erythrosin B. Measurements of the phosphorescence decay in the absence (nitrogen) and presence (air) of oxygen versus temperature provide estimates of the non-radiative decay rate for matrix-induced quenching (k _TS0) and oxygen quenching (k _Q[O₂]) of the triplet state. Since the oxygen quenching constant is the product of the oxygen solubility ([O₂]) and a term (k _Q) proportional to the oxygen diffusion coefficient, it is a measure of the oxygen permeability through the films. For all proteins except gelatin, Arrhenius plots of k _TS0 reveal a gradual increase of apparent activation energy across a broad temperature range starting at ∼50 °C; this suggests that there is a steady increase in the available modes of molecular motion with increasing temperature within the protein matrix. Arrhenius plots for k _Q[O₂] were linear for all proteins with activation energies ranging from 24 to 29 kJ/mol. The magnitude of the oxygen quenching constants varied in the different proteins; the rates were approximately 10-fold higher in α-La, β-Lg, and BSA than in 11S glycinin and gelatin. Although the rate of oxygen permeability was not directly affected by the increased mobility of the protein matrix, plots of k _Q[O₂] versus k _TS0 were linear over nearly three orders of magnitude in the protein films, suggesting that the matrix mobility plays a specific role in modulating oxygen permeability. This effect may reflect differences in matrix-free volume that directly influence both mobility and oxygen solubility.     

Fluidity of the lipid phase of bovine serum high density lipoprotein from fluorescence polarization measurements.

The microviscosity of the lipid phase of bovine serum high density lipoprotein was determined by fluorescence polarization measurements on a lipophilic probe (1,6-diphenyl-1,3,5-hexatriene) dissolved in the lipoprotein. At 25°C the average microviscosity was 6.1 ± 0.5 poise, and the activation energy calculated from a plot of log η versus $\text{1}\text{T}$ was 13±3Kcal/mole. A constant slope for the Arrhenius plot from 0 to 46°C indicated no apparent phase transitions in this temperature range.Comparison of the present results with reported microviscosity values for rat lymphocyte membranes and liposomes [Shinitzky and Inbar (1974) $\text{J. Mol. Biol. 85}$, 603] indicates a more rigid environment of the probe in the high density lipoprotein system fluidity of the lipid appears to be considerably decreased in the lipoprotein relative to organic solvent or oil solutions of lipids, probably as a result of the anisotropic environment of the probe, high total cholesterol, and presence of protein in these particles.     

Importance of membrane fluidity in the induction of alkaline phosphatase,a periplasmic enzyme,in Escherichiacoli

An unsaturated fatty acid auxotroph was supplemented with either elaidate or oleate. After derepression of alkaline phosphatase by phosphate limitation at 38°C, the cells were shifted to incubation at various temperatures. Arrhenius plots of the rate of enzyme induction gave a steeper negative slope in the temperature range from 30°C to 35°C with elaidate-supplemented cells than with oleate-supplemented cells. At 25°C the induction was arrested in the former cells, while it was continued at a considerable rate in the latter. The arrest was released upon shift-back to 38°C, and precursors convertible to the active enzyme were not accumulated during incubation at 25°C. There was no marked difference in slope of Arrhenius plots of the rate of bulk protein synthesis between both types of cells, and the slope was almost equal to that of the rate of enzyme induction in the oleate-supplemented cells. The rate of β-galactosidase induction in the elaidate cells showed a similar temperature dependence to that of bulk protein synthesis.     

Changes in Water Soluble Protein Profile of Antarctic Cyanobacterium Nostoc Commune under Temperature Downshift from 25° to 5 °C

The cyanobacterium Nostoc commune (Nostocales) is an isolate from the Schirmacher Oasis Antarctica. The cyanobacterium is psychrotropic in nature; and maintained in laboratory at 25?°C temperature, in unialgal form. Here, we studied the change in protein profile of water soluble proteins from exponentially growing N. commune upon downshift from its optimum growth temperature (25?°C) to a low temperature (5?°C). Experimental set up used to analyze the proteome were- a sudden shift to low temperature (i.e., cold shock), after short- (8?days) and long-term acclimation (7?weeks) to low temperature (5?°C). Cold-shock resulted in an increase in Low molecular weight proteins (LMWPs) with clouding of diffused proteins. Further increase in the duration of incubation period (short- and long-term acclimation) caused dissociation of proteins, indicated by NaCl (50–600mM) induced dissociation of proteins. That is, high molecular weight proteins (HMWPs) dissociated into LMWPs resulting in an increased number of protein bands. This was further confirmed by addition of LMWPs (≤10KDa) resulting in re-association of proteins into HMWPs. Hence, we report that the cold-induced synthesis of LMWPs (≤10kDa) is a strategy adopted by the N. commune to survive at low temperature of Antarctica.     

Sedimentation equilibrium studies of human chymotrypsin II and bovine -chymotrypsin

Sedimentation equilibrium experiments indicate that neither human chymotrypsin II nor bovine δ-chymotrypsin molecules undergo association in the pH range 3–5 where dimerization occurs with α-chymotrypsin. The weight-average molecular weights of human chymotrypsin II and δ-chymotrypsin in a pH 4.4 0.1 ionic strength buffer are 26,200 and 26,400, respectively, using the measured partial specific volumes of 0.722 and 0.727 ml/g at 25 °C. Number-average molecular weight calculations also support the presence of monomeric species at this pH. In the pH range 6–7.6 where sedimentation velocity studies have shown that δ-chymotrypsin associates at concentrations above 3 mg/ml, no association was observed for either the human chymotrypsin II or bovine δ-chymotrypsin in the sedimentation equilibrium experiments where protein concentrations were below 1.2 mg/ml. These studies provide additional evidence that human chymotrypsin II is similar to bovine δ-chymotrypsin.     

Effect of pH and Glucose on the Stability of α-lactalbumin

The objective of this study is to understand the influence of pH and effect of cosolvent (glucose) on the stabilization of bovine α-lactalbumin by using ultrasonic techniques. Values of density, ultrasonic velocity and viscosity were measured for bovine α-lactalbumin (5 mg/ml) dissolved in phosphate buffer (pH 2, 5, 7, 9 and 12) solutions mixed with and without the cosolvent at 30 °C. These measurements were used to calculate few thermo-acoustical parameters such as adiabatic compressibility, intermolecular free length, acoustic impedance, relaxation time, relative association constant, the partial apparent specific volume and the partial apparent specific adiabatic compressibility for the said systems. The obtained results revealed a strong comparison between the effects of acidic and alkaline pH values on protein denaturation, i.e., the acidic pH are instantaneous and are of less magnitude whereas alkaline pH are slower but sharper. Further the present study supports the fact that the presence of glucose stabilizes α-lactalbumin against denaturation due to pH variation, which may be due to the strengthening of non-covalent interactions and the steric exclusion effect.     

Heat Shock Response in Hypocotyl of Barley (Hordeum vulgare L.)

In vivo protein synthesis in barley (Hordeum vulgare L.) hypocotyl was maximum at 35°C and 40°C.HPLC analysis of soluble proteins showed 10 different types of proteins, out of which the peak corresponding to retention time 13.3 min was present at 25°C but was absent at 35°C and 40°C. Instead, another peak with retention time 13.7 min was noticed at 35°C and 40°C. Amino acid analysis showed that heat shock resulted in an increase in lysine and histidine and decrease in arginine. Heat shock also resulted in increase in peroxidase, protease and ribonuclease activity at 35°C and 37°C in comparison to 25°C. The incorporation of (3H)-uridine was significantly decreased at 37°C in comparison to 25°C.     

Prosopis chilensis is a plant highly tolerant to heat shock

At temperatures between 25 and 35°C, 100% of Prosopis chilensis seeds germinated within 24 h. At higher temperatures, the germination rate was reduced; at 50°C, seeds did not germinate. After germination at 25°C, the optimal temperature for seedling growth was 35°C and the seedlings did not grow at a temperature of 50°C. However, when germination was at 35°C, the optimal temperature for seedling growth was 40°C and some seedlings grew at 50°C, suggesting that thermotolerance was induced during seed germination at 35°C. Further thermotolerance can be induced in seedlings germinated at 35°C, by exposing them to 40°C for 2h. Under these conditions, seedlings exhibited increased growth rate at 45 and 50°C. Fluorography of SDS-polyacrylamide gel electrophoresis of the proteins synthesized and accumulated during 2 h at temperatures of 35, 40, 45 and 50°C in the presence of [³⁵S]methionine revealed the expression of 11 proteins not detectable at 35°C. Most of the proteins present at 35°C also increased in expression. The temperature for maximal expression of these proteins was 45°C.     

Development of a screening assay to measure the loss of antibacterial activity in the presence of proteins: its use in optimizing compound structure

An assay quantifying the loss of antibacterial potency of compounds, originally identified via target-based screening, in the presence of increasing albumin concentration was developed and used as a technique to measure potential association of compounds with proteins unrelated to their molecular target. Minimum inhibitory concentrations (MICs) of test compounds were measured against Staphylococcus aureus strain ATCC 6538 in the presence of 0-12 muM bovine serum albumin (BSA). The linear regression coefficient r(2) for the correlation between MIC and BSA concentration was >/= 0.9 for 49 and > 0.5 for 62 out of a total of 69 compounds tested. The slope of these correlations varied widely from < 1 to 99, suggesting that the loss of potency due to a given concentration of BSA could vary from compound to compound due to wide variation in the apparent stoichiometry for protein-ligand association. Follow-up experiments using additional proteins and a fatty acid, oleic acid, showed that this compound:BSA association was not protein specific, but was likely driven by hydrophobicity. The method described in this report can be used to optimize compound design and minimize this undesirable effect.     

The volume and compressibility changes of lysozyme associated with guanidinium chloride and pressure-assisted unfolding.

The apparent specific volumes and isentropic compressibilities of hen egg white lysozyme were measured in aqueous guanidinium chloride solutions at 25 degrees C by means of a vibrational densimeter and a sing-around ultrasonic velocimeter. Little transition attributable to a protein unfolding was detected in the partial specific volume, while the partial specific isentropic compressibility decreased slightly around the transition region. The pressure-assisted unfolding was also investigated in aqueous guanidinium chloride solutions by means of ultraviolet spectroscopy. Assuming a two-state transition model, it was found that the free energy change of unfolding depends almost linearly on pressure and the unfolding reaction is accompanied by a small decrease in volume. The compressibility behavior is in conflict with the notion that a protein structure is almost completely unfolded by guanidinium chloride and most of the amino acid residues in the protein interior are exposed to solvent. These results support the current view that globular proteins have some residual structures even in the unfolded state induced by a strong denaturant.