Temperature dependence of partial volumes of proteins

The change of the apparent partial specific volumes of egg albumin, bovine serum albumin, bovine methemoglobin, β-lactoglobulin, and lysozyme with temperature through the thermal transitions of the proteins have been measured with dilatometers. Four regions in the plot of the apparent partial specific volumes against temperature can be recognized: (1) linear sections extending from 25°C up to 45–50°C: (2) a decrease in slope between 50°C and 60°C; (3) a sharp increase in slope with increasing temperature coinciding with the appearance of heat coagulation of the protein and followed by (4) a decrease in the slope. The return of the protein samples to 25°C yields linear relations between the apparent partial specific volumes of the heat-denatured proteins and the decreasing temperature.     

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.     

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.     

Changes in the sarcoplasmic reticulum ATPase protein under denaturing conditions used for sodium dodecyl sulfate--polyacrylamide gel electrophoresis

The electrophoretic pattern of the sarcoplasmic reticulum (SR) ATPase protein was found to change, depending on the conditions used to denature the SR vesicles in sodium dodecyl sulfate (SDS), prior to SDS-polyacrylamide gel electrophoresis. A smearing of the gel pattern above the ATPase protein was observed when the SR vesicles were denatured at 100 °C in SDS, in the absence of β-mercaptoethanol (β-ME). Denaturation of the SR vesicles in SDS at 100 °C in the presence and the absence of β-ME reduced the amount of SR ATPase protein by half. More high-molecular-weight aggregates were formed in the presence than in the absence of β-ME. The other proteins of the SR as well as myofibrils and bovine serum albumin were found to be relatively unaffected by these treatments. It is concluded that, for the study of SR ATPase protein by SDS-polyacrylamide gel electrophoresis, denaturation at 100 °C should be avoided.     

Purification, characterization, and properties of beta-glucosidase enzymes from Sclerotium rolfsii

Four β-glucosidase enzymes were extensively purified from the culture filtrates of Sclerotium rolfsii and some of their physicochemical properties studied. All the enzymes showed a single protein band in sodium dodecyl sulfate-gel electrophoresis and in disc gel electrophoresis at pH 8.9 and 4.3. The purified β-glucosidases were free of endoglucanase (carboxymethyl cellulose viscosity-lowering activity). All the enzymes are glycoproteins and are composed of one polypeptide chain. The molecular weight of the four β-glucosidases varies between 90,000 and 107,000. The pH and temperature optima of the four β-glucosidases are 4.2 and 68 °C with p-nitrophenyl-β-d-glucoside and 4.5 and 65 °C with cellobiose as substrate. The isoelectric points for the enzymes are 4.10, 4.55, 5.10, and 5.55, respectively. The specific activities of the enzymes with cellobiose as substrate are 55, 78, 175, and 51 μmol glucose released per minute per milligram protein, respectively. The enzymes are inhibited by the reaction product glucose, and by glucono-δ-lactone and nojirimycin. A carboxylate group is implicated in the catalysis of β-glucosidase.     

Purification and properties of two enzymes from Dichomitus squalens which exhibit both cellobiohydrolase and xylanase activity

Two fractions (Ex I and II), exhibiting activity towards p-nitrophenyl β-cellobioside (pNPC) have been isolated by chromatofocusing of the proteins obtained from the supernatant solution of a cellulose-containing culture of the white-rot fungus Dichomitus squalens. They were further purified up to 16.0- and 14.2-fold by chromatography on Phenyl-Sepharose CL-4B and ion-exchange chromatography on DEAE-Trisacryl. Each purified enzyme gave a single peak for protein and activity on chromatography on Ultrogel AcA-54 and a single protein band in disc gel electrophoresis, in the absence and presence of sodium dodecyl sulphate, and on isoelectrofocusing. The mol. wts. of Ex I and II were 39,000 and 36,000, respectively, and their isoelectric points were 4.6 and 4.5, respectively. Maximum activity towards pNPC was shown at pH 5.0 and 60°, and each enzyme was stable over the pH range 4.0–8.0, and up to 70° and 60° for Ex I and II, respectively. The enzymes cleaved pNPC, released mainly cellobiose from cellulose, were especially active towards xylan and o-nitrophenyl β-d-xylopyranoside, and exhibited strong transglycosylating activities.     

Effect of Sorbed Water on the Thermal Stability of Soybean Protein

A soybean protein isolate (SPI), and its β-conglycinin and glycinin componets were obtained from defatted soybean flour by applying dissolution and precipitation based on the difference in their solubility depending on each isoelectric point. The purity evaluated by SDS–PAGE of the β-conglycinin and glycinin preparations was about 84% and 80%, respectively, resulting in a clear difference in the pH dependence on solubility. A BET plot derived from the water sorption isotherm at 25 °C showed that the amount of the monolayer adsorption of these preparations was about 6–9%, the value for the β-conglycinin preparation being about 1.5 times higher than that for the glycinin preparation. The β-conglycinin and glycinin preparations were respectively denatured at around 75 °C and 86 °C in the presence of excess water, whereas the denaturation temperature of both preparations was markedly increased by decreasing sorbed water content below 40%, corresponding well with the unfrozen water content.     

QUANTITATIVE AND QUALITATIVE ANALYSES OF PROTEIN SYNTHESIS DURING HEAT SHOCK IN THE MARINE DIATOM NITZSCHIA ALBA (BACILLARIOPHYCEAE)1

Protein synthesis in the diatom Nitzschia alba Lewin and Lewin was drastically altered when the cells were incubated at a supraoptimal temperaeture. Quantitatively, the overall protein synthesis was greatly suppressed as indicated by teh rate of [³⁵S] methionine incorporation. The extent of suppression of protein synthesis was proportional to the severity of the heat-shock treatment which was a combination of elevated temperature and treatment duration. The in viro synthesized proteins were also qualitativelty anlayzed by two-dimensional gel electrophoresis. Dependeing on the treatment condition, a set of heat-shock proteins (HSPs) were induced. They were best detected when the cells were subjected to shocks of 35°C for 60 min or 40°C for 10 min followed by a 60 min labelling at 30°C. The results revealed 16 HSps which had moluecular weights ranging from 24–94 kD and isoelectric points ranging from 5.50–7.10. Some of the HSps were identical in molelcular weights but with differeentr isoelectric points. The induction and accumulation of HSPs in Nitzschia alba were transitory. Prologned heat-shock treatments resulted in a complete cessation of protein syntehsis and no further induction of HSPs. In all aspects, the heat shock response of diatoms was similar to that in higher plants such as soybean, maize and tobacco but differenet from most animal systems.     

Protein Modifications in High Protein-Oil and Protein-Oil-Sugar Systems at Low Water Activity

Physicochemical and thermal properties of high protein systems during storage at 20 and 40 °C were investigated for 14 weeks. Component interactions of whey protein isolate (WPI)-olive oil (OO), WPI-sunflower oil (SO) (75:25), WPI-(glucose-fructose; G-F) (45:40), WPI-OO-(G-F), and WPI-SO-(G-F) (45:15:40) systems at low water contents during storage were derived from differential scanning calorimetry (DSC), colorimetric, water activity (a_w), reducing and nonreducing SDS-PAGE electrophoresis data. The degree of unsaturation of oil affected color (yellowness) and microstructure of the systems as well as variations in water migration and nonenzymatic browning kinetics (NEB) during storage. These effects were evident in the SO systems. All systems at 40 °C showed changes in protein conformation to those favoring hydrophobic interactions with oil. These systems showed decreased a_w, insolubilization, hardening as a result of carbonyl-amine polymerization and covalent cross-linking of proteins in the NEB. The DSC data showed a protein hydration transition for rehumidified-WPI, WPI-oil, WPI-sugar, and WPI-oil-sugar. The rehumidified-WPI and WPI-oil also showed a_w-dependent denaturation endotherms (irreversible transition) for α-lactalbumin and β-lactoglobulin at higher temperatures (T). The WPI-sugar and WPI-oil-sugar showed an exotherm for the browning reaction (irreversible transition) at T_onset ~ 90 °C. An exothermic protein hydration in the systems containing sugar was storage time-dependent, and indicated changes of protein conformation. The presence of oil in WPI-oil-sugar caused an increase in the glass transition of sugars during storage, especially for SO. The WPI-(G-F) and WPI-oil-(G-F) showed broadened glass transition during a reheating scan in DSC that was a result of polymerization in protein, oil, and sugar components mixture. Stability of high protein systems is dependent on hydration and reactions in both hydrophilic and hydrophobic phases.     

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 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.     

Solubilization and Identification of Hen Eggshell Membrane Proteins During Different Times of Chicken Embryo Development Using the Proteomic Approach

A fertilized chicken egg is a unit of life. During hatching, transport of nutrients, including calcium, have been reported from the egg components to the developing embryo. Calcium is mobilized from the eggshell with the involvement of Ca²⁺-binding proteins. In addition, other unknown proteins may also play some important roles during embryo developing process. Therefore identification and prediction of biological functions of eggshell membrane (ESM) proteins during chick embryo development was conducted by proteome analysis. Comparison of different lysis solutions indicated that the highest ability to extract ESM proteins could be obtained with 1 % sodium dodecyl sulfate in 5 mM Tris–HCl buffer pH 8.8 containing 0.1 % 2-mercaptoethanol. In this study fertilized Cornish chicken eggs were incubated at 37 °C in humidified incubators for up to 21 days. At selected times (days 1, 9, 15 and 21), samples were taken and the ESMs were carefully separated by hand, washed with distilled water, and air-dried at room temperature. The ESM proteins were then solubilized and analyzed by proteome analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with high performance liquid chromatography and mass spectrometry revealed 62 proteins in the ESM; only keratin is known ESM protein, 8 of which are egg white proteins and related while 53 others have not previously been reported. Some differences in the types of proteins and their molecular functions were noted in ESM at different incubation times. One protein which was present only at days 15 and 21 of egg incubation was identified as a calcium binding protein i.e. EGF like repeats and discoidin I like domain 3 (EDIL3 homologous protein).     

Nuclear protein redistribution in heat-shocked cells

An increase was observed in the total protein mass of nuclei isolated from Chinese hamster ovary cells heated at 45°C or 45.5°C. An increase in the fractional recovery of DNA polymerase α and β, and of DNA topoisomerase activity coincided with this increase in the protein mass of nuclei from heated cells. Nuclear protein mass which was soluble in 2.0 M NaCl decreased 0.5 fold, while DNA-associated and nuclear matrix-associated protein mass increased 2.2 and 3.4 fold, respectively. The results indicate that the increase in nuclear protein mass observed in nuclei from heated cells is due in part to an increased binding, or precipitation, of nuclear proteins onto the cell's DNA and nuclear matrix. © 1993 Wiley-Liss, Inc.     

Changes in Casein and Egg Albumin due to Reactions with Oxidizing Methyl Linoleate in Dehydrated Systems

Casein and egg albumin were allowed to react with methyl linoleate (ML) at a relative humidity (RH) of 0% or 80% at 50°C for 10 days (protein: ML= 1:0.2 or 1:1, w/w). Changes in the molecular sizes of the reacting proteins were examined by gel filtration and gel electrophoresis. Both proteins showed similar changes, whereas the reaction at RH 80% (protein: ML= 1:1) resulted in insolubilization because of polymerization. Changes in the amino acid residues of the reacting proteins were investigated after acid (6 n HC1) and enzymatic (pepsin-pancreatin, followed by aminopeptidase-prolidase) hydrolyses. Insignificant changes were observed in the amino acid composition of proteins reacted at RH 0%. After reaction at RH 80% (protein: ML =1:1), Lys, His and Met were the only amino acids affected. The percentage loss of these amino acids after acid hydrolysis was Lys (22%), His (41%), Met (9%) for casein and Lys (22%), His (31%), Met (1%) for egg albumin. This percentage loss after enzymatic hydrolysis was Lys (41%), His (49%), Met (94%) for casein and Lys (37%), His (42%), Met (88%) for egg albumin. Some differences between our results and other researchers were also discussed.     

Overexpression of a multifunctional β-glucosidase gene from thermophilic archaeon Sulfolobus solfataricus in transgenic tobacco could facilitate glucose release and its use as a reporter

The β-glucosidase, which hydrolyzes the β(1–4) glucosidic linkage of disaccharides, oligosaccharides and glucose-substituted molecules, has been used in many biotechnological applications. The current commercial source of β-glucosidase is mainly microbial fermentation. Plants have been developed as bioreactors to produce various kinds of proteins including β-glucosidase because of the potential low cost. Sulfolobus solfataricus is a thermoacidophilic archaeon that can grow optimally at high temperature, around 80 °C, and pH 2–4. We overexpressed the β-glucosidase gene from S. solfataricus in transgenic tobacco via Agrobacteria-mediated transformation. Three transgenic tobacco lines with β-glucosidase gene expression driven by the rbcS promoter were obtained, and the recombinant proteins were accumulated in chloroplasts, endoplasmic reticulum and vacuoles up to 1%, 0.6% and 0.3% of total soluble protein, respectively. By stacking the transgenes via crossing distinct transgenic events, the level of β-glucosidase in plants could further increase. The plant-expressed β-glucosidase had optimal activity at 80 °C and pH 5–6. In addition, the plant-expressed β-glucosidase showed high thermostability; on heat pre-treatment at 80 °C for 2 h, approximately 70% residual activity remained. Furthermore, wind-dried leaf tissues of transgenic plants showed good stability in short-term storage at room temperature, with β-glucosidase activity of about 80% still remaining after 1 week of storage as compared with fresh leaf. Furthermore, we demonstrated the possibility of using the archaebacterial β-glucosidase gene as a reporter in plants based on alternative β-galactosidase activity.

     

Heat shock response of Dictyostelium

In response to a shift from 22 to 30°C the relative rate of synthesis of a small number of proteins is dramatically increased in Dictyostelium discoideum. The cells neither grow nor develop at this temperature but die slowly with a half-life of 18 hr. The major protein synthesized in response to a heat shock to 30°C in either growing cells or developing cells has an apparent molecular weight of 70,000 (70K). An increase in the relative rate of synthesis of 70K can be seen as early as 20 min following heat shock. Synthesis of 70K remains high for 4 hr at 30°C and then decreases. Similar kinetics of 70K synthesis occur during recovery at 22°C following a 1-hr heat shock. RNA synthesis during the first half-hour of heat shock is essential for the high rate of 70K measured 2 hr later. By isoelectric focusing the 70K protein can be separated into two spots, one of which overlaps one of the major heat shock proteins of Drosophila melanogaster. The relative rate of synthesis of several other proteins (82K, 60K, 43K) increases less dramatically in Dictyostelium during heat shock at 30°C. A heat shock to 34°C results in rapid synthesis of these proteins but not of 70K. The relative rates of synthesis of most other proteins made at 22°C decreases, most notably that of actin. Synthesis of heat shock proteins at 30°C does not significantly affect viability at 30°C but dramatically prolongs the period of time the cells can survive at 34°C. Thus, 30°C appears to be a stasis condition for Dictyostelium which elicits a response essential for protection from lethal temperatures. The similarity of the heat shock response in Dictyostelium to that in Drosophila and vertebrate cells suggests that certain aspects of the response may be universal in eukaryotes.     

Mode of binding of cytochrome b5 to phospholipid bilayers in lamellar structure

X-ray diffraction studies were made on the multilamellar systems produced by incubation of phospholipid bilayers and the membrane protein, cytochrome b₅, or non-membrane proteins (albumin, ovalbumin and β-lactoglobulin A) at pH 8.1 in aqueous 5 mM CaCl₂ solutions.Detergent-extracted cytochrome b₅ (soluble aggregate) forms two types of lamellar phase with dipalmitoyl phosphatidylcholine bilayers, depending upon the incubation temperature. One type, which has a repeat distance of 114Å, is formed above 34°C, where the binding of cytochrome b₅ to the bilayers is hydrophobic. The other type, with a repeat distance of 153 Å, is formed below 34°C, where the binding is electrostatic. It is also suggested that cytochrome b₅ is monomeric in the former phase but remains aggregated in the latter phase.When dimyristoyl phosphatidylcholine is used, the boundary temperature for the two types shifts to 12°C. These boundary temperatures coincide with the thermal pretransition points of hydrated dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine, respectively.Trypsin-treated cytochrome b₅ (monomeric) and the three non-membrane proteins exhibit only binding of the electrostatic type to the bilayers, independently of the incubation temperature. The observed repeat distances suggest that in these cases two layers of protein molecules are incorporated between the bilayers.     

Antioxidative and Prooxidative Activity of Caffeic Acid toward H2O2-induced DNA Strand Breakage Dependent on the State of the Fe Ion in the Medium

Hen egg lysozyme–carboxymethyl dextran (HEL–CMD) conjugate was prepared by using water-soluble carbodiimide as a model protein-acidic polysaccharide conjugate for improving the protein function. An acid-amide bond between HEL and CMD was confirmed by SDS-PAGE, isoelectric focusing and IR spectra. The molar ratio of CMD to HEL in the conjugate was 1:1. The isoelectric point of the conjugate was 5.5–6.0, which is much lower than that of HEL. Spectroscopic studies suggested that the conformation around the Trp residue had not changed but the α-helix content had decreased to about 1/3 that for native HEL. The conjugate maintained about 60% of the enzymatic activity of native HEL at 40–60°C, while it was about 1.4 times as active as native HEL at 4°C and 80°C. The conjugate was more stable to proteolysis than native HEL. The denaturation temperature of the conjugate was about 73°C, which is almost the same as that of native HEL. However, the enthalpy for denaturation of the conjugate was about 1/3 that of native HEL, which corresponds to the decrease in α-helix content.     

The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide elongation rates

When Drosophila tissue culture cells are shifted from 25 to 36°C (heat shocked) the pre-existing mRNAs (25°C mRNAs) remain in the cytoplasm but their translation products are underrepresented relative to the induced heat shock proteins. Many of these undertranslated 25°C mRNAs are found in association with polysomes of similar size in heat-shocked and control cells. Furthermore, the messages encoding α-tubulin, β-tubulin, and actin are found associated with one-third to one-half as many total ribosomes in heat-shocked cells as in cells incubated at 25°C. Increased temperature should lead to increased output of protein per ribosome. However, the 25°C proteins are actually synthesized at less than 10% of 25°C levels in heat-shocked cells. Thus, the rates of both elongation and initiation of translation are significantly (15- to 30-fold) slower on 25°C mRNAs than they are on heat shock mRNAs in heat-shocked cells.     

Tyrosyl-phosphorylated proteins are involved in regulation of meiosis in the rat egg

Fertilization in invertebrates results in tyrosine (Tyr) phosphorylation of several egg proteins. However, the involvement of Tyr phosphorylation in mediating mammalian egg activation has not yet been investigated. Using an antibody specific for phosphotyrosine (P-Tyr), immunoblotting, and densitometric analysis, we found that maturation of the oocyte is accompanied by a generalized increase in the P-Tyr content of almost all egg proteins detected. After sperm penetration, 5 of the 17 protein bands detected demonstrated a small increase in their P-Tyr content, while at the pronuclear (PN) stage the signal was markedly reduced. Ionomycin emulated the changes observed at fertilization in most protein bands detected, demonstrating a small increase in their P-Tyr content within 15 min of exposure. Analysis of the involvement of the tyrosyl-phosphorylated, mitogen-activated protein (MAP) kinase during meiosis revealed comigration of the phosphotyrosyl bands with the protein and a good correlation with its enzyme activity. Maturation was accompanied by an increase in MAP kinase activity. The activity dropped partially after sperm penetration and furthermore later at the PN stage. A larger quantity accompanied by a more significant change in the P-Tyr content implies for extracellular regulated kinase (ERK) 2 being the dominant isoform present in the rat egg. Our results indicate that fertilization in mammals involves changes in activity of protein tyrosine kinases (PTKs) or in the balance between PTKs and protein tyrosine phosphatases. The single, ionomycin-induced Ca²⁺ rise is sufficient to imitate fertilization-induced changes in MAP kinase activity, as well as in tyrosine phosphorylation of other proteins within the egg. Mol. Reprod. Dev. 49:176–185, 1998. © 1998 Wiley-Liss, Inc.