Protein-cationic detergent interaction. Equilibrium dialysis study of the interaction of bovine serum albumin and other proteins with alkylpyridinium bromide.

The binding isotherms of native bovine serum albumin with cationic detergents, such as octyl, decyl, dodecyl and tetradecylpyridinium bromides were determined at pH 6.8 and 3.4 at 25 degrees C. The isotherms for dodecyl and tetradecylpyridinium bromides were also determined at 3 degrees C. The average number of detergent cations bound increased with increasing hydrocarbon chain length. At low detergent concentration the binding of all alkylpyridinium bromides was smaller at pH 3.4 than at pH 6.8. Dodecylpyridinium bromide was bound to native beta-lactoglobulin, aldolase, ovalbumin, haemoglobin, myoglobin, lysozyme, trypsin and ribonuclease at pH 6.8. No binding occurred to alpha-chymotrypsin and chymotrypsinogen. The free enthalpy change, --delta G degrees, calculated from intrinsic association constants K was determined.     

Protein-cationic detergent interaction. Fourier transform infrared and laser Raman spectroscopic studies on the interaction between proteins and dodecylpyridinium bromide.

Fourier transform infrared and laser Raman spectroscopies were used to study the effects of dodecylpyridinium bromide on the conformation of haemoglobin, myoglobin, bovine serum albumin, ribonuclease, ovalbumin, lysozyme, trypsin and beta-lactoglobulin in aqueous solution. Addition of the cationic detergent caused a decrease in alpha-helix conformation in highly helical proteins. At low detergent concentrations stabilization of beta-sheet conformation was observed.     

Protein-detergent interactions studied by capillary isotachophoresis

The interactions of sodium dodecyl sulphate with bovine serum albumin and ovalbumin have been studied by capillary isotachophoresis. This method makes it possible to determine very accurately the number of ligands bound to the high-affinity binding sites of the native protein. Bovine serum albumin was found to have seven high-affinity binding sites whereas ovalbumin in its native state was found to lack high-affinity binding sites for dodecyl sulphate.     

The binding of sodium dodecyl sulphate to bovine serum albumin at high binding ratios

The extent of binding of sodium dodecyl sulphate to bovine serum albumin at high binding ratios was investigated by gel filtration. The weight ratio of bound sodium dodecyl sulphate to bovine serum albumin increases with the NaCl concentration, and, except at low salt concentrations, with the concentration of sodium dodecyl sulphate. In the presence of 1.0g of sodium dodecyl sulphate/l, the binding ratio varied from 1.0 (at 0.04m-Na(+)) to 2.2 (at 0.44m-Na(+)). In the presence of 0.24m-Na(+), the binding ratio increased with sodium dodecyl sulphate concentration, from 0.9 (0.2g of sodium dodecyl sulphate/l) to 2.0 (5g of sodium dodecyl sulphate/l), at 26 degrees C, in a dilute sodium phosphate buffer. No significant dependence of the binding ratio upon temperature in the range 26-45 degrees C was observed. These results differ from those of Reynolds & Tanford (1970a) obtained by equilibrium dialysis.     

Characterization of a serum factor that decreases albumin mRNA in cultured hepatocytes

Summary When primary cultures of hepatocytes are exposed to media containing fetal bovine serum (FBS) there is a rapid decrease in levels of tissue-specific mRNAs such as albumin mRNA. We used Northern blot analysis to examine mRNA levels in cultured hepatocytes, and characterized the factor in FBS that significantly reduces the steady state albumin mRNA level. Neonatal bovine serum or serum derived from platelet-poor calf plasma proved as potent as did FBS, but commercial bovine serum albumin did not exhibit this inhibitory activity. Inhibitory activity of FBS was not removed by moderate heat treatment, dialysis, or extraction with organic solvents. However, incubation of FBS with a highly anionic detergent such as 0.1% sodium dodecyl sulfate orN-lauroyl sarcosine, followed by extensive dialysis, resulted in sera that did not inhibit expression of albumin mRNA. These sera supported cell attachment and seemed non-toxic toward the cells. Ammonium sulfate fractionation of FBS showed the activity was present in the 45 to 70% fraction, and trypsin digestion destroyed the inhibitory activity. Gel exclusion chromatography gave a molecular weight 60 000 to 70 000. Fractionation of serum proteins by DEAE-Sephacel or Cibacron blue-agarose showed enrichment for albumin in the most active fractions. Interestingly, metabolic labeling of secreted and cellular proteins with³⁵S-methionine and cysteine showed no significant difference between hepatocytes maintained for 2 days beforehand in serum-free or serum-supplemented media, and no difference between detergent-treated FBS and control FBS. Therefore, FBS contains a factor that causes a significant decrease in steady state levels of mRNA for albumin and other mRNAs of tissue specific function, but under these conditions albumin mRNA levels are not paralleled by synthesis of albumin or other proteins.     

A critical evaluation of the analysis of membrane proteins by polyacrylamide gel electrophoresis in the presence of dodecyl sulphate

Molecular weight determination by polyacrylamide gel electrophoresis in the presence of dodecyl sulphate assumes that (a) detergent treatment completely dissociates polypeptides from any non-covalent complex, (b) the binding of detergent is constant per unit length polypeptide chain and (c) dodecyl sulphate—polypeptide complexes all have the same conformation. The validity of these three assumptions with respect to membrane proteins is critically discussed.     

Electroblotting onto glass-fiber filter from an analytical isoelectrofocusing gel: a preparative method for isolating proteins for N-terminal microsequencing

A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% (v/v) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, beta-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (pI 5.6) glutathione S-transferase isolated from the chicken liver.     

Protein chromatography on adsorbents with hydrophobic and ionic groups. Chromatography of dodecyl sulphate-solubilized proteins of the human erythrocyte membrane on N-(3-carboxypropionyl)aminodecyl-sepharose.

Human erythrocyte 'ghosts' were solubilized in 0.5% (w/v) sodium dodecyl sulphate at pH 4.0(I = 0.012 mol/I). At a loading of 1-2 mg of protein/ml of column volume, all of membrane proteins were adsorbed to a column of CPAD [N-(3-carboxypropionyl)-aminodecyl]-Sepharose at pH 4.0 (I = 0-012 mol/1) and room temperature (22 degrees C). Many proteins were subsequently desorbed by raising the pH or by including sodium dodecyl sulphate continuously in the eluting buffer. Experiments with a series of adsorbents homologous with CPAD-Sepharose, in which the length of the hydrocarbon chain was varied, provided strong evidence of hydrophobic interactions, in addition to ionic interactions, in the binding of these proteins to CPAD-Sepharose. Elution with increasing-pH gradients at different concentrations of sodium dodecyl sulphate showed that glycophorin (the major sialoglycoprotein) was eluted in the void volume, at recoveries close to 100%, when the detergent concentration was greater than or equal to 0.3% (w/v). Protein E, the major protein, was desorbed late in the pH gradient even at a high (0.5%, w/v) concentration of the detergent, and was always incompletely desorbed, the maximum recovery recorded being 40%. Spectrin (the high-molecular-weight polypeptide pair) did not behave in a well-defined manner, and was found widely distributed among the effluent fractions under all the conditions that were tested.     

A microacalorimetric study of the interaction between trypsin and sodium n-dodecyl sulphate.

1. The binding of sodium n-dodecyl sulphate to trypsin and reduced trypsin has been measured by equilibrium dialysis at pH 3.5 and 5.5. 2. At pH 3.5 trypsin specifically binds surfactant at low concentration, at higher concentrations co-operative binding occurs. 3. Reduction of trypsin destroys the specific binding sites at pH 3.5. 4. At pH 5.5 both trypsin and reduced trypsin show only co-operative binding. 5. The interaction of sodium n-dodecyl sulphate with trypsin, reduced, inhibited, and thermally denatured trypsins has been studied by microcalorimetry at 25 degrees C. 6. The microcalorimetric measurements have been used to estimate enthalpy changes (deltaHd) on unfolding of trypsin; deltaHd = 82 +/- 5 kJ-mol-1 at pH 3.5 and 128 +/- 5 kJ-mol-1 at pH 5.5. 7. The unfolding of trypsin follows a different thermochemical pathway to that of reduced trypsin.     

A major transcriptional regulatory protein (ICP4) of herpes simplex virus type 1 is associated with purified virions

Herpes simplex virus type 1 was purified by density gradient centrifugation, and the virion-associated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By Western blot (immunoblot) analysis with an anti-ICP4 monospecific serum, the results indicated that ICP4, one of the five immediate-early proteins of herpes simplex virus type 1, was associated with the purified virions. To define the location of ICP4 within the virion, trypsin digestion experiments were performed. Purified virions were treated with trypsin in the presence or absence of detergent. The virus envelope appeared to protect ICP4 from the trypsin, since virus-associated ICP4 was sensitive to digestion only after detergent treatment. In addition, ICP4 remained associated with the virus particle when the virion-specific glycoproteins were removed after detergent treatment. Finally, ICP4 was not detected in purified preparations of type A and B capsids isolated from the nuclear fraction of virus-infected cells. The above-mentioned data suggest that detectable amounts of ICP4 are present within the tegument region of the virion.     

Spectroscopic determination of acetylcholine esterase–inhibitor complex: determination of conformational shifts of proteins

Changes in the absorbance spectrum of tetraphenylporphyrin sulfonate (TPPS) are observed that are unique for the proteins lysozyme, luciferase, apomyoglobin, myoglobin, gamma globulin, insulin, RNAase, phosphotransacetylase, papain, ovalbumin, bovine serum albumin (BSA), protamine sulfate, and polylysine. The absorbance spectrum of porphyrins is different for native compared with heat denatured RNAase. A unique absorbance wavelength red shift is observed with trypsin when trypsin inhibitor is present, indicating that porphyrins incorporated with proteins can detect conformational changes in the protein. The absorbance spectrum of the Soret band of TPPS undergoes bathochromic shifts upon addition of local anesthetics to acetylcholine esterase (AChE), suggesting that the absorbance spectrum of porphyrins can be used as a reporter of the presence of inhibitors of AChE by indicating conformational changes on binding of the inhibitor.     

Purification and Characterization of an Extracellular Acid Proteinase from the Ectomycorrhizal Fungus Hebeloma crustuliniforme

Hebeloma crustuliniforme produced an extracellular acid proteinase in a liquid medium containing bovine serum albumin as the sole nitrogen source. The proteinase was purified 26-fold with 20% activity recovery and was shown to have a molecular weight of 37,800 (as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and an isoelectric point of 4.8 +/- 0.2. The enzyme was most active at 50 degrees C and pH 2.5 against bovine serum albumin and was stable in the absence of substrates at temperatures up to 45 degrees C and pHs between 2.0 and 5.0. Pepstatin A, diazoacetyl-dl-norleucine methylester, metallic ions Fe and Fe, and phenolic acids severely inhibited the enzyme activity, while antipain, leupeptin, N-alpha-p-tosyl-l-lysine chloromethyl ketone, and trypsin inhibitor inhibited the activity moderately. The proteinase hydrolyzed bovine serum albumin and cytochrome c rapidly compared with casein and azocasein but failed to hydrolyze any of the low-molecular-weight peptide derivatives tested.     

Characterization of a proteolytic-enzyme inhibitor with allergenic activity. Multiple functions of a parasite-derived protein.

1. A trypsin inhibitor from the tick Boophilus microplus was purified by ion-exchange chromatography and gel filtration. 2. It is pure by the criteria of constant specific activity on gel filtration and by electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate. 3. The protein undergoes reversible polymerization, dissociating at low pH. 4. The apparent molecular weight measured by electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate is 18,500. 5. Inhibition of trypsin occurs by formation of a 1 :1 molar complex. 6. Chymotrypsin is also inhibited, though the dissociation constant of the complex formed is larger than with trypsin. The protein possesses independent sites for the inhibition of chymotrypsin and trypsin. 7. The inhibitor preparation gives an immediate hypersensitivity reaction on intradermal injection into cattle that have been exposed to the tick. The allergenic activity is due to the inhibitor protein itself and not to contaminating material, since the two activities were not separated during purification or in two subsequent affinity-chromatography procedures. 8. The hypersensitivity reaction is a true immunological response, since it is found in almost all cattle that have been exposed to the tick, but not in unexposed animals. In addition, passive cutaneous anaphylaxis can be demonstrated with serum from exposed, but not from unexposed, animals.     

Further evidence for the heterogeneity of serum albumin

Albumin samples from three species (avian, bovine and human) were electrophoresed on gradient polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS-PAGE). The resulting electrophoregram from each sample of serum albumin investigated showed multiple protein bands of a wide range of molecular weights. All seven samples of human serum albumin were found, using gel immunodiffusion, to be contaminated with other proteins. All but one sample was contaminated with proteins such as haptoglobin, alpha 1-glycoprotein, alpha 1-trypsin inhibitor, and prealbumin. This contamination accounts for part of the heterogeneity of these samples. Immunoblots, where the proteins were transferred to nitrocellulose and incubated with antisera, gave a better demonstration of the heterogeneity than Coomassie Blue staining and the immunoblotting procedure appeared to be more sensitive than the gel immunodiffusion technique. The heterogeneity of serum albumin demonstrated by the former technique included that of the monomer which was shown to be contaminated with antithrombin III. The commercial samples of human serum albumin, claimed as pure, were found to vary greatly in their tryptophan content, which also indicated heterogeneity. Heat treatment of human serum albumin with 1% SDS, followed by chromatography on agarose, removed the protein contaminants and with it the tryptophan. The presence of tryptophan in human serum albumin, therefore, indicated the presence of impurities.     

Characterization of the apolipoprotein B polypeptide of human plasma low density lipoprotein in detergent and denaturation solutions.

Apolipoprotein B, the polypeptide moiety of human serum low density lipoprotein, is subject to degradation (as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) both in the intact particle and after delipidation. Protease inhibitors, sodium azide, and nitrogen saturation did not influence the rate or degree of degradation. Lipid-free apolipoprotein B prepared by gel exclusion chromatography in sodium dodecyl sulfate bound a limited number of detergent molecules (up to 300) in monomeric sodium dodecyl sulfate solutions; circular dichroic spectra of this complex were similar to spectra of the intact lipoprotein. Near the critical micelle concentrations, a large, cooperative increase in detergent binding occurred, accompanied by circular dichroic changes indicating increased alpha helicity. By sucrose density centrifugation, lysopalmitoyl phosphatidylcholine could be substituted for the anionic detergent; about 300 mol of lysolipid were bound to the polypeptide. Replacement of detergent with guanidine hydrochloride by dialysis produced a soluble polypeptide with no ordered structure at denaturant concentrations above 7 M. At lower guanidine hydrochloride concentrations, structural elements were regained in a broad, reversible transition. It appears that apolipoprotein B is an easily degraded polypeptide with regions resembling water-soluble proteins but other regions which interact with lipid (or synthetic amphiphiles) and produce an overall insolubility in aqueous solution in the absence of amphiphilic ligands.     

Purification and properties of buffalo serum albumin

Based on a detailed study of the solubility of serum albumin, a procedure for its purification by selective ammonium sulphate precipitation has been developed. Using buffalo serum, first extraneous proteins were precipitated by making the serum 2.26 M saturated with ammonium sulphate at pH 7.0 and then albumin was precipitated from the supernatant at 1.9 M ammonium sulphate concentration at pH 4.2. The overall yield of serum albumin thus isolated was about 55% with a purity of 97%. The protein preparation gave a single nearly symmetrical peak on Sephadex G-100 column and virtually a single band on polyacrylamide gel electrophoresis in the presence and absence of SDS. Buffalo serum albumin has a molecular weight of 69,000 Da. The hydrodynamic properties such as Stoke's radius (3.70 nm), diffusion coefficient (6.03 X 10(-7) cm2/s) and frictional ratio (1.36) obtained by analytical gel chromatography, bilirubin binding characteristics and its interaction with anti-bovine serum albumin antiserum suggest that buffalo serum albumin is very similar to BSA in its molecular properties.     

On the interaction of alpha2-plasmin inhibitor and proteases. Evidence for the formation of a covalent crosslinkage and non-covalent weak bondings between the inhibitor and proteases.

alpha2-plasmin inhibitor is a proteinase inhibitor in plasma which efficiently inhibits the lysis of fibrin clots induced by plasminogen activator. The nature of the binding of the inhibitor to trypsin or plasmin was studied by the chemical treatment of the enzyme-inhibitor complex with 7.5 M hydrazine at pH 10.0. With the hydrazine treatment, the complexes were degraded to proteins corresponding to the respective enzyme and inhibitor moieties. These results indicate that the covalent bond between the inhibitor and the enzymes is a carboxylic ester. The binding reaction of the inhibitor to active site-modified trypsin was also studied. The inhibitor formed complexes with anhydrotrypsin and carboxyamidomethylated trypsin. The complexes were dissociated in the presence of 1% sodium dodecyl sulfate, to the individual components: the respective enzyme and inhibitor moieties. The inhibitor, however, did not form a complex with diisopropylphosphorylated trypsin regardless of the presence or absence of the denaturing reagent. These results suggest the contribution of non-covalent interactions to the complex formation between the inhibitor and native enzymes.     

Wheat-germ aspartate transcarbamoylase. The effect of ligands on the inactivation of the enzyme by trypsin and denaturing agents

In the absence of added ligands aspartate transcarbamoylase (EC 2.1.3.2) from wheat germ is inactivated fairly rapidly by trypsin, by heat (60 degrees C), by highly alkaline conditions (pH11.3) and by sodium dodecyl sulphate. Addition of UMP alone, at low concentrations, decreases the rate of inactivation by each of these agents significantly. Carbamoyl phosphate alone does not alter the rate of inactivation by trypsin and by the detergent, but it antagonizes the effect of UMP in protecting the enzyme against these agents. These results have been interpreted to mean that two conformational states are reversibly accessible to the enzyme, namely an easily inactivated state favoured in the presence of carbamoyl phosphate and a more resistant state favoured in the presence of UMP. In the absence of ligands the enzyme is in the easily inactivated conformation. At very high concentrations l-aspartate also protects the enzyme but to a smaller extent than UMP. Some implications of these results are discussed.     

Proteolysis in heat-stressed HeLa cells. Stabilization of ubiquitin correlates with the loss of proline endopeptidase

When intact HeLa cells were incubated at 45 degrees C, there was progressive inactivation of proline endopeptidase. Rapid loss of the enzyme did not occur in extracts maintained at 45 degrees C. Since Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no decrease in the immunoreactive 70-kDa proline endopeptidase band, its in vivo disappearance apparently results from irreversible denaturation or modification. Loss of proline endopeptidase activity was paralleled by reduced degradation of injected ubiquitin and bovine serum albumin. In contrast, proteolysis of injected lysozyme or pancreatic trypsin inhibitor was barely affected. Electrophoretic analysis of ubiquitin or bovine serum albumin retrieved from heated HeLa cells showed that the injected proteins were intact. Thus, the presence of proline endopeptidase appears to be required for initial cleavage of these two substrates, but it has not been shown that the enzyme is directly responsible. Selective stabilization of a subset of the injected proteins does, however, demonstrate the existence of distinct proteolytic pathways in HeLa cytosol.     

5-methyltetrahydrofolate: synthesis and utilization in normal and SV40-transformed BHK-21 cells.

Dodecyl sulfate complexes of two soluble proteins, serum albumin and fumarase, have been “renatured” with large excesses of the nonionic detergent Triton X-100. The resulting complexes, essentially free of dodecyl sulfate, differ in their sedimentation properties relative to the native protein and in their interaction with Triton X-100. Albumin molecules refold to a form binding only very small amounts of Triton and have a sedimentation coefficient similar to that of the non-denatured protein. On the other hand, refolded fumarase molecules have a lower sedimentation coefficient than that of the native enzyme and bind up to 1.06 mg of Triton/mg protein. It is postulated that the fumarase molecule has been turned “inside-out” by the dodecyl sulfate/Triton treatment, and the implications of such large conformational changes for protein transport across membranes are discussed.