SULFHYDRYL AND DISULFIDE GROUPS OF PROTEINS : II. THE RELATION BETWEEN NUMBER OFSH AND S-S GROUPS AND QUANTITY OF INSOLUBLE PROTEIN IN DENATURATION AND IN REVERSAL OF DENATURATION

1. In native egg albumin no SH groups are detectable, whereas in completely coagulated albumin as many groups are detectable as are found in the hydrolyzed protein. In egg albumin partially coagulated by heat the soluble fraction contains no detectable groups, and the insoluble fraction contains the number found after hydrolysis. 2. In the reversal of denaturation of serum albumin, when insoluble protein regains its solubility, S-S groups which have been detectable in the denatured protein, disappear. 3. When egg albumin coagulates at an air-water interface, all the SH groups in the molecule become detectable. 4. In egg albumin coagulated by irradiation with ultraviolet light, the same number of SH groups are detectable as in albumin coagulated by a typical denaturing agent. 5. When serum albumin is denatured by urea, there is no evidence that S-S groups appear before the protein loses its solubility. 6. Protein denaturation is a definite chemical reaction: different quantitative methods agree in estimates of the extent of denaturation, and the same changes are observed in the protein when it is denatured by different agents. A protein molecule is either native or denatured. The denaturation of some proteins can be reversed.     

THE EFFECT OF WATER CONTENT UPON THE RATE OF HEAT DENATURATION OF CRYSTALLIZABLE EGG ALBUMIN

1. The denaturation rate of partially dried crystallizable egg albumin is greatly decreased by decreasing its water content. 2. The temperature of denaturation, defined as the temperature at which half of the protein becomes insoluble in distilled water after a definite time of heating, is a linear function of the relative humidity with which the protein is in equilibrium. 3. By applying the Arrhenius equation it is shown that the rate of heat denaturation at a given temperature is an exponential function of the relative humidity. 4. The application of the observed relations to the analysis of the mechanism of thermal death of microorganisms is suggested. 5. The water content of native and heat-denatured egg albumin is determined as a function of the relative humidity of water vapor. It is shown that the heat-denatured modification takes up approximately 80 per cent as much water at all relative humidities as does native egg albumin.     

SULFHYDRYL GROUPS IN FILMS OF EGG ALBUMIN

1. The same number of SH groups reduces ferricyanide in surface films of egg albumin as in albumin denatured by urea, guanidine hydrochloride, Duponol, or heat, provided the ferricyanide reacts with films while they still are at the surface and with the denatured proteins while the denaturing agent (urea, heat, etc.) is present. 2. The SH groups of a suspension of egg albumin made by clumping together many surface films react with ferricyanide in the same sluggish and incomplete manner as do the groups in egg albumin denatured by concentrated urea when the urea is diluted or in albumin denatured by heat when the solution is allowed to cool off. 3. The known change in configuration of the egg albumin molecule when it forms part of a surface film explains why SH groups in the film react with ferricyanide whereas those in native egg albumin do not. In the native egg albumin molecule groups in the interior are inaccessible to certain reagents. A film is so thin that there are no inaccessible groups. 4. Because of the marked resemblance in the properties of egg albumin in surface films and of egg albumin after denaturation by the recognized denaturing agents, it may be supposed that the same fundamental change takes place in denaturation as in film formation—indeed, that film formation is one of the numerous examples of denaturation. This would mean that in general the SH groups of denatured egg albumin reduce ferricyanide and react with certain other reagents because they are no longer inaccessible to these reagents.     

THE DENATURATION OF EGG ALBUMIN BY ULTRA-VIOLET RADIATION

The coagulation of isoelectric egg albumin solutions, on exposure to ultraviolet radiation, involves three distinct processes, (1) the light denaturation of the albumin molecule, (2) a reaction between the light denatured molecule and water which may be similar to heat denaturation but occurs at a lower temperature, and (3), the flocculation of the denatured molecules to form a coagulum. The light denaturation is unimolecular, independent of temperature, and occurs over a wide pH range. The reaction between the light denatured molecule and water has a temperature coefficient of 10⁺ and occurs rapidly at 40°C., a temperature at which heat denaturation is inappreciable.     

Dielectric study of the urea denaturation of delipidated and relipidated bovine serum albumin

Dielectric relaxation and viscosity measurements were performed on delipidated and relipidated samples of bovine serum albumin (BSA) at urea concentrations between O and 6M. By the combined interpretation of these two hydrodynamic methods the characterization of conformational changes of the molecule during urea denaturation is possible. The denaturation of delipidated BSA results from two mechanisms. The first one is a slow, time-dependent elongation of the molecule; the second one is a rapid swelling which becomes most pronounced at urea concentrations higher than 4M. For relipidated albumin, the slow elongation mechanism occurs but the presence of fatty acids protects the protein aganist molecular swelling. In both cases these conformational changes are accompanied by an increased disymmetry of charge repartition and a concomitant increase of the dipole moment. From these results it follows that lipidated albumin (as occurs under physiological conditions) is less sensitive to denaturation than delipidated albumin.     

SULFHYDRYL GROUPS OF EGG ALBUMIN IN DIFFERENT DENATURING AGENTS

1. The reaction between ferricyanide and egg albumin in solutions of urea, guanidine hydrochloride, and Duponol has been investigated. 2. In neutral medium ferricyanide oxidizes all the SH groups of egg albumin that give a color reaction with nitroprusside. In neutral medium ferricyanide appears to react only with the SH groups of egg albumin. The quantity of ferrocyanide formed can accordingly be considered the equivalent of the number of SH groups in egg albumin detectable with nitroprusside. 3. In solutions of urea, guanidine hydrochloride, and Duponol sufficiently concentrated so that all the egg albumin present is denatured, the same number of SH groups are found—equivalent to a cysteine content of 0.96 per cent. 4. In denaturation of egg albumin loss of solubility (solubility not in presence of the denaturing agent, but solubility examined in water at the isoelectric point) and appearance of reactive SH groups are integral parts of the same process. As denaturation proceeds in urea, SH groups are liberated only in the egg albumin with altered solubility and in this albumin the maximum number of SH groups is liberated. In a molecule of egg albumin either all of its SH groups that give a test with nitroprusside are liberated or none of them are.     

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.     

Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.     

Urea equilibrium unfolding of the major core protein of the retrovirus feline immunodeficiency virus and its tryptophan mutants

Circular dichroism and fluorescence spectroscopy have been employed to study the urea unfolding mechanism of a recombinant form of the major core protein of feline immunodeficiency virus (FIV-rp24) and its native tryptophan mutants. The equilibrium denaturation curves indicate the existence of two transitions. The first unfolding transition most likely reflects the denaturation of the carboxy-terminal region of FIV-rp24. Consequently, the second transition, where the changes in fluorescence are produced, should reflect the denaturation of the amino-terminal region. If the intermediate observed upon urea denaturation is an on-pathway species, the data described herein can reflect the sequential and independent loss of structure of the two domains that this type of proteins possesses.     

The denaturation of ribonuclease A by combinations of urea and salt denaturants.

When urea is added to ribonuclease A that has already been denatured by salt (CaCl₂, LiClO₄ or LiCl were used), a second co-operative transition occurs, supporting the previous demonstration that these salts cause only partial denaturation. Also we have studied the effect of the salts on the urea denaturation, and the effect of urea on the salt denaturation. At low concentrations urea makes the salt transitions occur at lower concentrations, but at higher concentrations it changes the transition so that the completely disordered protein found in urea is produced by the salt. At low concentrations the salts actually stabilize the protein against denaturation by urea, but at higher concentrations they destabilize it. The data are presented in “phase diagrams” which are found to be very useful for such three-component systems.     

SOME FACTORS WHICH INFLUENCE THE OXIDATION OF SULFHYDRYL GROUPS

1. Cyanide inhibits the oxidation of the SH groups of cysteine and denatured egg albumin by the uric acid reagent. 2. At pH 4.8 cysteine is oxidized by the uric acid reagent and by ferricyanide in the presence but not in the absence of added copper sulfate. 3. In neutral solution, the uric acid reagent oxidizes the SH groups of denatured egg albumin in the presence of urea but not in the presence of alkyl sulfate or in the absence of denaturing agents. 4. Ferricyanide oxidizes the SH groups of neutral denatured egg albumin even in the presence of alkyl sulfate or, if precautions are taken to avoid aggregation, in the absence of denaturing agents. 5. In acid solution, ferricyanide does not oxidize the SH groups of denatured egg albumin completely. The oxidation is more complete, however, in the presence of urea than in the presence of alkyl sulfate, and more complete in the presence of guanidine hydrochloride than in the presence of urea. 6. The uric acid reagent which does not oxidize the SH groups of neutral denatured but unhydrolyzed egg albumin in the absence of denaturing agents does, under the same conditions, oxidize the SH groups of egg albumin partially hydrolyzed by pepsin. 7. At pH 4.8 in alkyl sulfate solution ferricyanide oxidizes the SH groups of digested egg albumin more completely than the SH groups of denatured but undigested egg albumin.     

The measurement of insoluble proteins using a modified Bradford assay

A technique for determining the amount of thermally denatured, insoluble protein is described. The assay has been validated using four globular proteins, bovine serum albumin, beta-lactoglobulin, lysozyme, and ovalbumin. It consists of a resolubilization protocol, using 8 M urea and 5% 2-mercaptoethanol, linked to the Bradford dye binding assay. The resolubilization protocol was carried out at 100 degrees C to enable complete recovery of all insoluble proteins. Beta-Lactoglobulin resolubilization was completed after heating for 1 min, whereas samples of bovine serum albumin, lysozyme, and ovalbumin required heating for 1.5 min. The assay can measure protein concentrations as small as 10 micrograms, typically with standard deviations of 3%, thus comparing favorably with the standard Bradford assay. Other types of denaturation, such as chemical denaturation causing subsequent insolubility, may be studied with this technique providing that there is no interference with the Bradford assay.     

THE SULFHYDRYL GROUPS OF EGG ALBUMIN

1. 1 cc. of 0.001 M ferricyanide, tetrathionate, or p-chloromercuribenzoate is required to abolish the SH groups of 10 mg. of denatured egg albumin in guanidine hydrochloride or Duponol PC solution. Both the nitroprusside test and the ferricyanide reduction test are used to show that the SH groups have been abolished. 2. 1 cc. of 0.001 M ferrocyanide is formed when ferricyanide is added to 10 mg. of denatured egg albumin in neutral guanidine hydrochloride or urea solution. The amount of ferricyanide reduced to ferrocyanide by the SH groups of the denatured egg albumin is, within wide limits, independent of the ferricyanide concentration. 3. Ferricyanide and p-chloromercuribenzoate react more rapidly than tetrathionate with the SH groups of denatured egg albumin in both guanidine hydrochloride solution and in Duponol PC solution. 4. Cyanide inhibits the oxidation of the SH groups of denatured egg albumin by ferricyanide. 5. Some samples of guanidine hydrochloride contain impurities which bring about the abolition of SH groups of denatured egg albumin and so interfere with the SH titration and the nitroprusside test. This interference can be diminished by using especially purified guanidine hydrochloride, adding the titrating agent before the protein has been allowed to stand in guanidine hydrochloride solution, and carrying out the nitroprusside test in the presence of a small amount of cyanide. 6. The SH groups of egg albumin can be abolished by reaction of the native form of the protein with iodine. It is possible to oxidize all the SH groups with iodine without oxidizing many of the SH groups beyond the S-S stage and without converting many tyrosine groups into di-iodotyrosine groups. 7. p-chloromercuribenzoate combines with native egg albumin either not at all or much more loosely than it combines with the SH groups of denatured egg albumin or of cysteine. 8. The compound of mercuribenzoate and SH, like the compound of aldehyde and SH and like the SH in native egg albumin, does not give a nitroprusside test or reduce ferricyanide but does reduce iodine.     

Use of domain specific ligands to study urea-induced unfolding of bovine serum albumin

Urea-induced structural transitions in different domains of bovine serum albumin (BSA) were studied fluorometrically using domain specific ligands; chloroform, bilirubin, and diazepam. Urea denaturation of BSA showed a two-step, three-state transition with the accumulation of an intermediate around 4.8-5.2 M urea. During first transition (0-5.0 M urea), a continuous decrease (starting from 1.0 M urea) in diazepam (a ligand for domain III) binding and a late (from 3.0 M urea onward) decrease in chloroform (a ligand primarily for domain I) binding suggested major conformational changes in domain III and partial but significant loss of native conformation in domain I prior to intermediate formation. Absence of any decrease in bilirubin (a ligand for domain II) binding up to 4.5 M urea indicated non-involvement of domain II in the unfolding of BSA in this region. However, decrease in bilirubin binding during second transition reflected the unfolding of domain II and its separation from domain I.     

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.     

THE COMBINATION OF ENZYME AND SUBSTRATE : I. A METHOD FOR THE QUANTITATIVE DETERMINATION OF PEPSIN. II. THE EFFECT OF THE HYDROGEN ION CONCENTRATION.

1. A quantitative method for the determination of pepsin is described depending on the change in conductivity of a digesting egg albumin solution. 2. The combination of pepsin with an insoluble substrate has been followed by this method. 3. The amount of pepsin removed from solution by a given weight of substrate is independent of the size of the particles of the substrate. 4. There is an optimum zone of hydrogen ion concentration for the combination of enzyme and substrate corresponding to the optimum for digestion. 5. It is suggested that the pepsin combines largely or entirely with the ionized protein.     

Applying the increase in rate constants of cooperative proteolysis to the determination of transition curves of protein denaturation

The transition curves of soybean glycinin egg lysozyme, bovine serum albumin (BSA) denaturation under the action of increasing urea concentration were monitored by determination of the increase in rate constants of cooperative enzymatic proteolysis. The results were compared with those obtained using intrinsic fluorescence and the number of accessible tyrosine. It was shown that the determination of the changes of proteolysis rate permits to disclose conformation changes not detected by other methods.     

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.     

Differences in the processes of beta-lactoglobulin cold and heat denaturations.

The changes in beta-lactoglobulin upon cold and heat denaturation were studied by scanning calorimetry, CD, and NMR spectroscopy. It is shown that, in the presence of urea, these processes of beta-lactoglobulin denaturation below and above 308 K are accompanied by different structural and thermodynamic changes. Analysis of the NOE spectra of beta-lactoglobulin shows that changes in the spin diffusion of beta-lactoglobulin after disruption of the unique tertiary structure upon cold denaturation are much more substantial than those upon heat denaturation. In cold denatured beta-lactoglobulin, the network of residual interactions in hydrophobic and hydrophilic regions of the molecules is more extensive than after heat denaturation. This suggests that upon cold- and heat-induced unfolding, the molecule undergoes different structural rearrangements, passing through different denaturation intermediates. From this point of view, cold denaturation can be considered to be a two stage process with a stable intermediate. A similar equilibrium intermediate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However, these processes differ in cooperativity, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption with cooling is accompanied by cooperative changes in heat capacity, whereas with heating the heat capacity changes only gradually. Considering the sigmoidal shape of the heat capacity change an extended heat absorption peak, we propose that the intermediate state is stabilized by enthalpic interactions.     

Calorimetric study of the thermal denaturation of beta-lactoglobulin in the presence of urea and phosphate ions]

The denaturation of beta-lactoglobulin in solution with different content of urea and phosphates has been studied calorimetrically. It has been shown that the increase of phosphate ion concentration in solution leads to an increase of beta-lactoglobulin stability, while increase of urea concentration leads to an opposite effect. The variation of these components in solution practically does not influence the value of the heat capacity increment of beta-lactoglobulin in the considered temperature region. Accordingly the denaturation enthalpy is a linear function of temperature whose slope does not differ for solution with urea concentration less than 4.4 M. However, the absolute value of denaturation enthalpy in these solutions at corresponding temperatures differs significantly due to the heat effect of additional urea solvation during transition to the denatured state. The latter leads to a decrease of the overall denaturation enthalpy and, as a result, a shift of the enthalpy plot to higher temperatures providing conditions for studying the thermodynamic and structural characteristics of the molecule in the cold denatured-state.