Interaction of alcohols with proteins

The kinetics of denaturation of egg albumin have been determined for methanol, ethanol, propanol, and butanol. The reactions are first order in respect to protein but between 11th and 18th order for the alcohols. The denaturation reaction is characterized by a large temperature coefficient with little or no dependence on pH. There is a marked change of pH when proteins are denatured. A series of eight proteins has been studied. There is surprisingly little difference in susceptibility to alcohol denaturation between the various proteins. Methanol, ethanol, propanol, and butanol are strongly bound to egg albumin—butanol being the most strongly bound. The binding of alcohol is probably accompanied by protein dehydration. The polyhydric alcohols' behavior is much different. These alcohols do not denature proteins and the protein is hydrated. Sucrose produces the greatest degree of hydration.     

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.     

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.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : III. VISCOSITY.

1. Gelatin solutions have a high viscosity which in the case of freshly prepared solutions varies under the influence of the hydrogen ion concentration in a similar way as the swelling, the osmotic pressure, and the electromotive forces. Solutions of crystalline egg albumin have under the same conditions a comparatively low viscosity which is practically independent of the pH (above 1.0). This difference in the viscosities of solutions of the two proteins seems to be connected with the fact that solutions of gelatin have a tendency to set to a Jelly while solutions of crystalline egg albumin show no such tendency at low temperature and pH above 1.0. 2. The formulæ for viscosity demand that the difference in the order of magnitude of the viscosity of the two proteins should correspond to a difference in the relative volume occupied by equal masses of the two proteins in the same volume of solution. It is generally assumed that these variations of volume of dissolved proteins are due to the hydration of the isolated protein ions, but if this view were correct the influence of pH on viscosity should be the same in the case of solutions of gelatin, of amino-acids, and of crystalline egg albumin, which, however, is not true. 3. Suspensions of powdered gelatin in water were prepared and it was found, first, that the viscosity of these suspensions is a little higher than that of gelatin solutions of the same concentration, second, that the pH influences the viscosity of these suspensions similarly as the viscosity of freshly prepared gelatin solutions, and third, that the volume occupied by the gelatin in the suspension varies similarly as the viscosity which agrees with the theories of viscosity. It is shown that this influence of the pH on the volume occupied by the gelatin granules in suspension is due to the existence of a Donnan equilibrium between the granules and the surrounding solution.     

THE REACTIONS OF DENATURED EGG ALBUMIN WITH FERRICYANIDE

The following facts have been established experimentally. 1. In the presence of the synthetic detergent, Duponol PC, there is a definite reaction between dilute ferricyanide and denatured egg albumin. 0.001 mM of ferrocyanide is formed by the oxidation of 10 mg. of denatured egg albumin despite considerable variation in the time, temperature, and pH of the reaction and in the concentration of ferricyanide. 2. If the concentration of ferricyanide is sufficiently high, then the reaction between ferricyanide and denatured egg albumin in Duponol solution is indefinite. More ferrocyanide is formed the longer the time of reaction, the higher the temperature, the more alkaline the solution, and the higher the concentration of ferricyanide. 3. Denatured egg albumin which has been treated with formaldehyde or iodoacetamide, both of which abolish the SH groups of cysteine, does not reduce dilute ferricyanide in Duponol PC solution. 4. Cysteine is the only amino acid which is known to have a definite reaction with ferricyanide or which is known to react with dilute ferricyanide at all. The cysteine-free proteins which have been tried do not reduce dilute ferricyanide in Duponol PC solution. 5. Concentrated ferricyanide oxidizes cystine, tyrosine, and tryptophane and proteins which contain these amino acids but not cysteine. The reactions are indefinite, more ferrocyanide being formed, the higher the temperature and the concentration of ferricyanide. 6. The amount of ferrocyanide formed from denatured egg albumin and a given amount of ferricyanide is less in the absence than in the presence of Duponol PC. 7. The amount of ferrocyanide formed when denatured egg albumin reacts with ferricyanide in the absence of Duponol PC depends on the temperature and ferricyanide concentration throughout the whole range of ferricyanide concentrations, even in the low range of ferricyanide concentrations in which ferricyanide does not react with amino adds other than cysteine. The foregoing results have led to the following conclusions which, however, have not been definitely proven. 1. The definite reaction between denatured egg albumin in Duponol PC solution and dilute ferricyanide is a reaction with SH groups whereas the indefinite reactions with concentrated ferricyanide involve other groups. 2. The SH groups of denatured egg albumin in the absence of Duponol PC react with iodoacetamide and concentrated ferricyanide but they do not all react rapidly with dilute ferricyanide. 3. Duponol PC lowers the ferricyanide concentration at which the SH groups of denatured egg albumin react with ferricyanide. The SH groups of denatured egg albumin, however, are free and accessible even in the absence of Duponol PC.     

A statistical mechanical study of helix-coil transition in concentrated solutions of polypeptides and proteins

The helix–coil transition in polypeptides was studied by methods of statistical mechanics, taking account of interaction between “melted” amide groups through hydrogen bonds. The statistical sums are calculated in the explicit form for two limit cases: (1) the dilute solution when the main contribution is given by collisions of two particles; (2) “condensation” when contacting macromolecules form a united aggregate. In the first case the transition enthalpy was shown to decrease linearly when the concentration increases, while at the appropriate choice of theoretical parameters, melting temperature and range are almost independent of the concentration. In the second case the structural transition parameters were shown to be independent of the concentration (the saturation effect). These results agree with the experimental data on synthetic polypeptides reported by other authors and with data on some globular proteins (serum and egg albumin) reported in this paper.     

ION SERIES AND THE PHYSICAL PROPERTIES OF PROTEINS. I

1. This paper contains experiments on the influence of acids and alkalies on the osmotic pressure of solutions of crystalline egg albumin and of gelatin, and on the viscosity of solutions of gelatin. 2. It was found in all cases that there is no difference in the effects of HCl, HBr, HNO₃, acetic, mono-, di-, and trichloracetic, succinic, tartaric, citric, and phosphoric acids upon these physical properties when the solutions of the protein with these different acids have the same pH and the same concentration of originally isoelectric protein. 3. It was possible to show that in all the protein-acid salts named the anion in combination with the protein is monovalent. 4. The strong dibasic acid H₂SO₄ forms protein-acid salts with a divalent anion SO₄ and the solutions of protein sulfate have an osmotic pressure and a viscosity of only half or less than that of a protein chloride solution of the same pH and the same concentration of originally isoelectric protein. Oxalic acid behaves essentially like a weak dibasic acid though it seems that a small part of the acid combines with the protein in the form of divalent anions. 5. It was found that the osmotic pressure and viscosity of solutions of Li, Na, K, and NH₄ salts of a protein are the same at the same pH and the same concentration of originally isoelectric protein. 6. Ca(OH)₂ and Ba(OH)₂ form salts with proteins in which the cation is divalent and the osmotic pressure and viscosity of solutions of these two metal proteinates are only one-half or less than half of that of Na proteinate of the same pH and the same concentration of originally isoelectric gelatin. 7. These results exclude the possibility of expressing the effect of different acids and alkalies on the osmotic pressure of solutions of gelatin and egg albumin and on the viscosity of solutions of gelatin in the form of ion series. The different results of former workers were probably chiefly due to the fact that the effects of acids and alkalies on these proteins were compared for the same quantity of acid and alkali instead of for the same pH.     

THE EFFECT OF PURE PROTEIN SOLUTIONS AND OF BLOOD SERUM ON THE DIFFUSIBILITY OF CALCIUM

1. A comparative study has been made of the diffusibility of calcium in solutions of crystalline egg albumin, serum globulin, and human blood serum. 2. In all three of these solutions, at pH 7.4, molal Ca concentrations within the membrane are greater than the calcium concentrations in the outside solutions, quite in accordance with the Donnan theory. 3. At pH 7.4, the ratio of See PDF for Structure varies directly with the protein concentration whether the solution be one of egg albumin, serum globulin, or blood serum. This is also in accordance with the Donnan theory. 4. On the acid side of the isoelectric point of the proteins, the concentration of Ca outside becomes greater than the concentration in the solution of blood serum or pure protein, as is demanded by the Donnan theory. 5. The magnitude of the Ca ratios on the alkaline and acid sides of the isoelectric points is probably the resultant of the Donnan equilibrium and the formation of complex Ca-protein ions. Northrop and Kunitz have shown the probability of the existence of such ions in the case of Zn⁺⁺, K⁺, and Li⁺, where satisfactory electrodes have been developed for E.M.F. measurements.     

Static Light Scattering From Concentrated Protein Solutions II: Experimental Test of Theory for Protein Mixtures and Weakly Self-Associating Proteins

Using an experimental technique recently developed in this laboratory (Fernández C. and A. P. Minton. 2008. Anal. Biochem. 381:254-257), the Rayleigh light scattering of solutions of bovine serum albumin, hen egg white ovalbumin, hen egg white ovomucoid, and binary mixtures of these three proteins was measured as a function of concentration at concentrations up to 125 g/L. The measured concentration dependence of scattering of both pure proteins and binary mixtures is accounted for nearly quantitatively by an effective hard particle model (Minton A. P. 2007. Biophys. J. 93:1321-1328) in which each protein species is represented by an equivalent hard sphere, the size of which is determined by the nature of repulsive interactions between like molecules under a given set of experimental conditions. The light scattering of solutions of chymotrypsin A was measured as a function of concentration at concentrations up to 70 g/L at pH 4.1, 5.4, and 7.2. At each pH, the measured concentration dependence is accounted for quantitatively by an effective hard particle model, according to which monomeric protein may self-associate to form an equilibrium dimer and, depending upon pH, an equilibrium pentamer or hexamer.     

The interaction of albumin and fatty-acid-binding protein with membranes: oleic acid dissociation

Bovine serum albumin or fatty-acid-binding protein rapidly lose oleic acid when incubated in the presence of dimyristoyl lecithin liposomes. The phenomenon is dependent on vesicle concentration and no measurable quantities of protein are found associated with liposomes. Upon gel filtration on Sepharose CL-2B of incubated mixtures of microsomes containing [1-14C] oleic acid and albumin or fatty-acid-binding protein, association of fatty acid with the soluble proteins could be demonstrated. Both albumin and fatty-acid-binding protein stimulated the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes. These results indicate that albumin is more effective in the binding of oleic acid than fatty-acid-binding protein, which allows a selective oleic acid dissociation during its interaction with membranes.     

THE REACTIONS OF IODINE AND IODOACETAMIDE WITH NATIVE EGG ALBUMIN

The following experimental results have been obtained. 1. Native egg albumin treated with iodine and then denatured no longer gives a nitroprusside test or reduces dilute ferricyanide in neutral Duponol PC solution. 2. More iodine is needed to abolish the ferricyanide reduction if the reaction between native egg albumin and iodine is carried out at pH 6.8 than if the reaction is carried out at pH 3.2. At pH 6.8 iodine reacts with tyrosine as well as with cysteine. 3. Cysteine and tryptophane are the only amino acids with reducing groups which are known to react with dilute iodine at pH 3.2 The reducing power of cysteine is abolished by the reaction with iodine, whereas the reducing power of tryptophane remains intact. Pepsin and chymotrypsinogen which contain tryptophane but not cysteine, do not react at all with dilute iodine at pH 3.2. 4. Native egg albumin treated with iodoacetamide at pH 9.0 and then denatured by Duponol PC reduces only 60 per cent as much dilute ferricyanide as egg albumin which has not been treated with iodoacetamide. 5. The SH group is the only protein reducing group which is known to react with iodoacetamide. The simplest explanation of the new observation that the SH groups of egg albumin can be modified by reactions with the native form of the protein is that the native egg albumin has free and accessible but relatively unreactive SH groups which can react with iodine and iodoacetamide despite the fact that they do not react with ferricyanide, porphyrindin, or nitroprusside. Preliminary experiments suggested by the results with egg albumin indicate that the tobacco mosaic virus is modified by iodine at pH 2.8 without being inactivated and that the tobacco mosaic and rabbit papilloma viruses are not inactivated by iodoacetamide at pH 8.0.     

Comparison of biotin binding protein of pregnant rat serum with rat serum albumin

The purified biotin binding protein of pregnant rat serum was shown to be immunologically similar to rat serum albumin as assessed by a sensitive radioimmunoassay. In radioimmunoassay for rat biotin binding protein, the binding of [¹²⁵I] rat biotin binding protein to anti-chicken egg yolk biotin binding protein antibodies was displaced by both rat serum (10–100 nl) and purified rat serum albumin (0.1–10 ng). Similarly, in radioimmunoassay for rat serum albumin the binding of [¹²⁵I] rat serum albumin to either anti-rat serum albumin antibodies or anti-chicken egg yolk biotin binding protein antibodies was displaced by unlabelled rat biotin binding protein at comparable concentration range (0·5–10 ng). Significant fractions of radioiodinated rat biotin binding protein and rat serum albumin bound to antibodies to chicken egg yolk biotin binding protein. In immature rats, the circulating half-lives of rat biotin binding protein and rat serum albumin were determined to be 12 and 17 h respectively. The rat biotin binding protein and rat serum albumin were analysed by techniques that exploit their physicochemical properties. They displayed similar electrophoretic mobilities in alkaline as well as denaturing sodium dodecyl sulphate-polyacrylamide gels. However, in nonequilibrium pH gradient polyacrylamide gel electrophoresis, they resolved clearly. In two-dimensional tryptic peptide map analysis, the two proteins showed similarities as well as significant differences in the relative distribution patterns of their iodopeptides. These results showed that the primary structure of rat biotin binding protein and rat serum albumin were different in finer details despite the fact that they shared significant immunological cross-reactivity.     

Minimum and optimum physiological doses of egg albumin and mutton protein in newly weaned rats

Using diets with a mounting egg albumin and mutton protein concentration, the authors determined the optimum physiological doses, which are identical with the maximum of linearity of the given parameters, from changes in body nitrogen, body weight and body water. The regression equations of the lines from these parameters were used to determine the minimum physiological doses for newly weaned rats. The optimum and minimum physiological doses of the amino acids in the relevant dietary proteins were also determined by analysing the source for its nitrogen content and other components and for the amino acid spectrum. The optimum dose of egg albumin protein for newly weaned rats was 1.69 g/day (on a 15% protein diet) and of mutton protein 2.56 g/day (on a 12.5% protein diet), with daily amounts of 60 mg methionine, 93 mg phenylalanine, 112 mg valine and 60 mg tyrosine for both proteins. The minimum doses of egg albumin and mutton protein, determined from changes in body nitrogen were 282 mg and 213 mg/day respectively [methionine 10.4 (4.7) mg/day, phenylalanine 16.1 (7.5) mg/day, valine 19.1 (9.2) mg/day and tyrosine 9.8 (5.2) mg/day)].     

THE NATURE OF THE IONIZABLE GROUPS IN PROTEINS

Analysis of the experimental titration curves shows that gelatin contains acid groups with dissociation indices at pH 2.9 to 3.5 corresponding quantitatively with the content in dicarboxylic amino acids; and that the acidic group at pH 9.4 in egg albumin agrees with the amount of tyrosine. The amounts of histidine and lysine present in both these proteins agree quantitatively with basic groups at pH 6.1 and pH 10.4 to 10.6, respectively. However, the quantity of the arginine group (pH 8.1) in these proteins is considerably less than the amount of arginine found on hydrolysis. This deficiency is compensated (quantitatively with gelatin and approximately with egg albumin) by a basic group at pH 4.6. The structure of this "4.6 group" should be similar to aniline and cytosine in consisting of an amino group on a conjugated unsaturated (perhaps cyclic) system. It would appear that the 4.6 group is disrupted on hydrolysis, producing arginine, and may be referred to as "prearginine." The presence of prearginine in proteins, instead of the full amount of arginine, has an important effect on the properties. Otherwise the isoelectric point of gelatin would be 8.0 (instead of 4.7) and of egg albumin 6.6 (instead of 4.8), and the titration curves would be quite different in shape between pH 4 and 10. Deamination of gelatin produces no decrease in prearginine, arginine, or histidine groups, but removes nearly all of the lysine group.     

THE ADSORPTION OF EGG ALBUMIN ON COLLODION MEMBRANES

An experimental study has been made of the adsorption of purified egg albumin, from aqueous solution, on collodion membranes. At protein concentrations of 4 to 7 per cent apparent saturation values were obtained which resembled closely the results obtained with gelatin, showing a maximum at pH 5.0 and lower values on either side of this region. Over large ranges of protein concentration, however, the curves for the adsorption from solutions removed in either direction from the isoelectric point exhibited a different shape from the hyperbola obtained in the neighborhood of pH 5.0. The addition of NaCl to solutions on the acid side tended to obliterate the effect of the pH difference; on the alkaline side it greatly increased the adsorption. The adsorption at 25° was about twice as great as that at 1°. The theory of the swelling of submicroscopic particles, advanced to account for the adsorption behavior of gelatin, is not sufficient to explain the results obtained with egg albumin. It is suggested that the effect is related to alterations in the forces causing the retention of the protein on the membranes.     

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.     

Preparation of Water-soluble and Stable Chlorophylls and β-Carotene by Forming Complexes with Egg Albumin

Chlorophyll- and β-carotene-egg albumin complexes were prepared. As these pigment-protein complexes were soluble in water, the chlorophylls and β-carotene were endowed with a water-soluble character. The molecular numbers of pigments incorporated into the complexes were increased by using proteins treated with charcoal, and additionally by using chemically modified proteins, acetylated or carbamoylated egg albumin. When compared with the stabilities of these pigments in detergent or in an organic solvent, the chlorophyll in the complexes was stable against photobleaching and for a wide range of pH values (pH 5 ~ 10). β-Carotene in the egg albumin complexes was relatively stable under UV irradiation.     

Thermal transitions of myosin and its helical fragments. I. Shifts in proton equilibria accompanying unfolding.

The thermal transitions of myosin and its helical fragments have been studied with pH as the observable. Heating unbuffered solutions of these proteins near their pI values causes an abrupt rise in pH at a characteristic temperature (the "melting temperature," Tm) which is due to structural changes within the protein. Since the pH shift turns out to be insensitive to the degree of protein aggregation, we have obtained acceptable melting curves even under conditions where the protein coagulates during melting. The melting profiles and Tm vlaues of myosin, myosin rod, and light meromyosin have been found to be remarkably similar (Tm equal to 40 plus or minus 1 degree, 0.5 M KCl, pH 5.9). Proton binding which occurs during melting coincides with the unfolding of a section of myosin rod. Taken in the context of other studies, the proton binding is thought to occur near the "hinge region."     

Study of heat denaturation of human serum albumin in water-alcohol and water-salt solutions in the presence of organic ligands

The method of differential scanning microcalorimetry was used to show a decrease in heat stability of serum albumin in the presence of aliphatic alcohols. In aqueous-alcohol media, the melting temperature, denaturation transition enthalpy were decreased, and the protein intermolecular aggregation enhanced. When the alcohol concentration in aqueous solution was elevated, the number of epsilon-amino groups of lysine residues in human serum albumin exposed to the solvent rose from 6-7 in aqueous solution to maximum 20 groups in the aqueous-alcohol solution, respectively. The elevation of ionic strength also induced an increase in the number of exposed lysine residues and was accompanied by an enhancement of protein aggregation. The modification of six amino groups by pyridoxal phosphate or three by glucose in the initial albumin stabilized the protein incubated at 65 degrees-70 degrees C both in the aqueous-alcohol media. At the given concentration and temperature the native protein was denatured and fully aggregated. Aliphatic alcohols displaced fatty acids from the binding sites on the molecule of serum albumin, which resulted in a change in the number of peaks of the melting curve.     

Thermal transitions of myosin and its helical fragments. II. Solvent-induced variations in conformational stability.

The melting behavior of myosin and myosin rod has been studied over the pH range 5.4-7.0, in 0.5 M KCl. Both proteins exhibit almost identical T-m values, which increase from about 37 to 43 degrees as the pH is elevated through the range of study, T-m follows a sigmoidal dependence upon pH, and the inflection point occurs near pH 6.5. The influence of salt concentration on T-m was studied, by the variation of KCl from 0.15 to 2.92 M. With an increasing KCl concentration, both proteins exhibit similar, sigmoidal declines in T-m from about 44 to 34 degrees. Under all conditions of pH and ionic strength studied, melting is accompanied by an absorption of H+ by the protein. The concentration of the protein, the age of the preparation, and the presence of divalent metal ions fail to exert a significant effect on the T-m values obtained by our methods. The effects of salt concentration and pH on the thermal stability of myosin and myosin rod are discussed in terms of the location of the melting process within the myosin molecule. Myosin is shown to possess several of the requisite features for energy transduction via a proton-coupling mechanism. These features provide a framework for investigating some aspects of the molecular basis of muscle contraction within the context of the sliding filament model.