The temperature dependence of the surface tension of aqueous solutions of plasma proteins.

The surface behavior of aqueous solutions of fibrinogen, transferrin, gamma-globulin and albumin at the liquid-gas interface has been investigated by a modified Wilhelmy technique. The temperature dependence of the surface tension was studied over a temperature range of 20--80 degrees C and a pH range of 2--12. Most pronounced conformational changes of fibrinogen with this technique were found in physiological conditions: 35--45 degrees C and pH 7--8. A conformational change was found for gamma-globulin and transferrin solutions, but at a higher temperature and less pronounced than fibrinogen. Albumin did not undergo conformational transitions to a significant extent.     

Effect of N-linked glycosylation on the aspartic proteinase porcine pepsin expressed from Pichia pastoris

A study was undertaken to examine the effects of N-linked glycosylation on the structure-function of porcine pepsin. The N-linked motif was incorporated into four sites (two on the N-terminal domain and two on the C-terminal domain), and the recombinant protein expressed using Pichia pastoris. All four N-linked recombinants exhibited similar secondary and tertiary structure to nonglycosylated pepsin, that is, wild type. Similar K(m) values were observed, but catalytic efficiencies were approximately one-third for all mutants compared with the wild type; however, substrate specificity was not altered. Activation of pepsinogen to pepsin occurred between pH 1.0 to 4.0 for wild-type pepsin, whereas the glycosylated recombinants activated over a wider range, pH 1.0 to 6.0. Glycosylation on the C-terminal domain exhibited similar pH activity profiles to nonglycosylated pepsin, and glycosylation on the N-domain resulted in a change in activity profile. Overall, glycosylation on the C-domain led to a more global stabilization of the structure, which translated into enzymatic stability, whereas on the N-domain, an increase in structural stability had little effect on enzymatic stability. Finally, glycosylation on the flexible loop region also appeared to increase the overall structural stability of the protein compared with wild type. It is postulated that the presence of the carbohydrate residues added rigidity to the protein structure by reducing conformational mobility of the protein, thereby increasing the structural stability of the protein.     

Effect of intersubunit interaction in horse liver alcohol dehydrogenase on the kinetics of ethanol oxidation]

The kinetics of enzymatic oxidation of ethanol in the presence of alcohol dehydrogenase within a wide range of ethanol and NAD concentrations (pH 6.0--11.5) were studied. It was shown that high concentrations of ethanol (greater than 0.7--5 mM, depending on pH) and NAD (greater than 0.4--0.8 mM) activate alcohol dehydrogenase from horse liver within the pH range of 6.0--7.9. A mechanism of activation based on negative cooperativity of ADH subunits for binding of ethanol and NAD was proposed. The catalytic and Michaelis constants for alcohol dehydrogenase were calculated from ethanol and NAD at all pH values studied. The changes resulting from the subunit cooperativity were revealed. The nature of ionogenic groups of alcohol dehydrogenase, which affect the formation of complexes between the enzyme and NAD and ethanol, and the rate constants for catalytic oxidation of ethanol was assumed. The biological significance of the enzyme capacity for activation by high concentrations of ethanol within the physiological range of pH in the blood under excessive use of alcohol is discussed.     

The enzymatic digestion of elastin at acidic pH

The enzymatic degradation of insoluble elastin has been studied at several pH values using purified pepsin and cathepsin D, and neutrophil extracts. Pepsin degraded elastin throughout the pH range of 1.2-4.0 with the optimum pH below 2.0. Molecular sieve chromatography and gel electrophoresis indicated that a spectrum of molecular weight degradation products was produced. The degradation by pepsin was inhibited by sodium dodecyl sulfate (SDS), NaCl and pepstatin. Cathepsin D, which, like pepsin, degrades hemoglobin at acid pH and is inhibited by pepstatin, had no activity against insoluble elastin in the pH range of 3.2-7.2. Extracts of neutrophils degraded elastin above pH 4.0. The pH profile of elastin degradation by neutrophil extracts generally followed that of purified human leukocyte elastase. Our results suggest that during alimentation or pulmonary aspiration of gastric contents, extracellular elastin may be digested by gastric juice at acid pH. Inflammatory cells would not appear to be capable of contributing to such actions until local pH approaches neutrality. Cathepsin D, a major constituent of inflammatory cells, does not digest all types of connective tissue proteins.     

Investigation on enzymatic degradation of γ-polyglutamic acid from Bacillus subtilis NX-2

The preparation of γ-polyglutamic acid (γ-PGA) from Bacillus subtilis NX-2 has been previously investigated, and its depolymerization during the batch culture was studied in this paper. The results suggested that the γ-PGA depolymerase was present and active extracellularly in the culture. The ywtD gene from B. subtilis NX-2, encoding the γ-PGA depolymerase was cloned and expressed in Escherichia coli. The YwtD protein was purified by metal-chelating affinity chromatography. YwtD was proved to be an endo-hydrolase enzyme and exhibited a remarkable activity in γ-PGA degradation at a wide range of temperature (30–40 °C) and pH (5.0–8.0). On an optimal condition of 30 °C and pH 5.0, an efficient γ-PGA enzymatic degradation was achieved. The molecular weight of γ-PGA could be reduced within the range of 1000–20 kDa and the polydispersity also decreased as a function of depolymerization time. Therefore, a controllable degradation of γ-PGA could be available by enzymatic depolymerization.     

Enzymatic desorption of layer-by-layer assembled multilayer films and effects on the release of encapsulated indomethacin microcrystals

Polyelectrolyte multilayer films were prepared through layer-by-layer (LbL) self-assembly of chitosan (CHI) and pyrene labeled poly(2-acrylamido-2-methylpropanesulfonic acid) (APy). After incubation in an enzyme pepsin solution, multilayer films were partially destroyed as detected by a decrease in fluorescence intensity due to enzymatic degradation of CHI and desorption of APy. The multilayer desorption rate was the highest at pH 4.0. Increasing temperature from 20 degrees C to 60 degrees C accelerated desorption. The enzymatic desorption was also observed from microcapsule walls made of CHI/alginate (ALG) multilayer films directly deposited on indomethacin (IDM) microcrystals by LbL self-assembly. After pepsin erosion, the IDM release from the microcapsule monitored by UV absorbance was obviously accelerated due to desorption. The influence of incubation time, pH, and temperature of the pepsin solution on the IDM release was investigated. The release rate was the fastest after incubation in the pepsin solution at pH 4.0 due to the highest activity of pepsin. Increasing incubation temperature from 20 degrees C to 60 degrees C, however, slowed down the release rate, which was considered to be due to the formation of more perfect and compact multilayer films through the chain rearrangement at higher temperatures. The CHI/ALG multilayer film was found to maintain its barrier function to the IDM diffusion even after 6-h incubation in the pepsin solution.     

Polyethyleneimine phosphate and citrate systems act like pseudo polyampholytes as a starting method to isolate pepsin

The aqueous solution behaviour of polyethyleneimine (a cationic synthetic polymer) in the presence of anions (such as citrate and phosphate) was studied by means of turbidimetry. The variation of the absorbance at 420 nm of dilute mixture with pH, the polymer concentration and the ionic strength were examined. The mixture of polyethyleneinine citrate or polyethyleneinine phosphate behaves as a pseudo polyampholyte with an isoelectric point of 5.5 and 6.2 for phosphate and citrate respectively and a precipitation pH range between 3.5 and 8.0. Pepsin was completely precipitated with the polymer anion complex within this pH interval. Citrate showed a better precipitation effect than phosphate did. The precipitate was reversibly dissolved in NaCl (for concentrations higher than 0.2 M) and pepsin kept its biological activity. Studies of pepsin thermal stability (by differential scanning calorimetry) revealed that the polyethyleneimine presence increased the enzyme denaturation temperature. The circular dichroism spectrum of pepsin showed a non-significant loss of secondary and tertiary enzyme structure by the polyethyleneimine. However, the polymer presence increased the biological activity of pepsin.     

Conformation of pepsin and pepsinogen: some aspects of the role of tyrosine residues and the 1-44 segment of pepsinogen on conformational stability

Conformational changes induced in pepsin and pepsinogen by iodination of tyrosine residues and the possible role of lysine residues on conformational stability of pepsinogen are investigated by circular dichroism (CD) studies in solution. At low degrees of iodination (6 I/molecule) the pepsin molecule denatured, with complete loss of β-structure at pH 5.5. Pepsinogen showed greater resistance to conformational change on iodination (10 I/molecule) and about 30% of its ordered structure is retained. In the aromatic region, the tyrosyl CD bands of iodinated pepsin decreased in intensity, indicating a change in the environment of tyrosine residues. A comparison with the CD spectra of expanded structures of pepsin in 6 m guanidine hydrochloride or alkaline solutions (pH 9.75) indicated retention of a significant amount of tertiary structure in iodinated pepsin. Changes in tertiary structures were marginal on iodination of pepsinogen. Less than 1% (residue moles) of poly-l-lysine, a known inhibitor, was found to destabilize the secondary and tertiary structure of pepsin at pH 6.75, although the lysine-rich 1–44 segment of pepsinogen tends to stabilize the conformation of the pepsin chain. This seems to suggest that the inhibitory effects of polylysine on pepsin occur by a mechanism different from that of the activity-limiting effect of the lysine-rich 1–44 segment of pepsinogen.     

ISOLATION,CRYSTALLIZATION, AND PROPERTIES OF SWINE PEPSINOGEN

1. A method is described for the preparation of pepsinogen from swine gastric mucosae which consists of extraction and fractional precipitation with ammonium sulfate solutions followed by two precipitations with a copper hydroxide reagent under particular conditions. Crystallization as very thin needles takes place at 10°C., pH 5.0 and from 0.4 saturated ammonium sulfate solution containing 3–5 mg. protein nitrogen per milliliter. 2. Solubility measurements, fractional recrystallization, and fractionation experiments based on separation after partial heat or alkali denaturation and after partial reversal of heat or alkali denaturation failed to reveal the presence of any protein impurity. 3. The properties of the enzymatically inactive pepsinogen were studied and compared with the properties of crystalline pepsin. The properties of pepsinogen which are similar to those of pepsin are: molecular weight, absorption spectrum, tyrosine-tryptophane content, and elementary analysis. The properties in which they differ are: enzymatic activity, crystalline form, amino nitrogen, titration curve, pH stability range, specific optical rotation, isoelectric point, and the reversibility of heat or alkali denaturation. 4. Conversion of pepsinogen into pepsin at pH 4.6 was found to be autocatalytic; i.e., the pepsin formed catalyzes the reaction. Conversion of pepsinogen into pepsin is accompanied by the splitting off of a portion of the molecule containing 15–20 per cent of the pepsinogen nitrogen.     

The effect of pre-incubation on trypsin kinetics at low pH.

A possible source of discrepancy between kinetic and spectroscopic studies of the active site ionizations in the enzyme trypsin (EC 3.4.21.4) could arise if a slow pH-dependent conformational change affected the rates at low pH. No such effect is observed within the time range of 1 min- 3 h when pre-incubation of trypsin at pH 2.0 or at pH 6.9 precedes the enzymatic hydrolysis of Nalpha-carbobenzoxy-L-lysine-p-nitrophenyl ester. The deacylation rate of this hydrolysis depends on a single pKa on the enzyme between pH 3 and pH 7.     

Studies on the Mechanism of Activation of Pepsinogen

The mechanism of activation of pepsinogen was studied. It was found that no peptide bond cleavage occurred in the molecule of denatured pepsinogen at pH 2. It was inferred from this that a specific secondary and tertiary structure is formed in the molecule of pepsinogen in acid and that it might be necessary for the hydrolysis of the peptide bond. From the circular dichroism studies on pepsinogen and pepsin, it was found that there is a conformational change in the molecule of pepsinogen at pH 4.3~4.5 and that this change is followed by a gradual formation of pepsin.     

Ovalbumin digestion by human pepsins 1, 3 and 5.

1. Of the three major human pepsins, pepsin 1 has greater proteolytic activity towards ovalbumin than has pepsin 3. Pepsin 5 has low activity towards this substrate. 2. Proteolytic pH-activity curves show only on pH maximum, about pH 1.4 for pepsin 1, pH 1.4--1.5 for pepsin 3 and pH 1.2--1.4 for pepsin 5. The curve for pepsin 3 has a shoulder between pH 2.4 and 3.4. 3. The rate of digestion of ovalbumin by pepsin 1 is approximately three times slower than are those of bovine haemoglobin or human globin. 4. The results suggest that there may be a physiological advantage in having more than one pepsin.     

Acid pH-induced conformational changes in bovine liver rhodanese.

The enzyme rhodanese is greatly stabilized in the range pH 4-6, and samples at pH 5 are fully active after several days at 23 degrees C. This is very different from results at pH greater than 7, where there is significant loss of activity within 1 h. A pH-dependent conformational change occurs below pH 4 in a transition centered around pH 3.25 that leads slowly to inactive rhodanese at pH 3 (t 1/2 = 22 min at pH3). The inactive rhodanese can be reactivated by incubation under conditions required for detergent-assisted refolding of denatured rhodanese. The inactive enzyme at pH 3 has the maximum of its intrinsic fluorescence spectrum shifted to 345 nm from 335 nm, which is characteristic of native rhodanese at pH greater than 4. At pH 3, rhodanese shows increased exposure of organized hydrophobic surfaces as measured by 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid binding. The secondary structure is maintained over the entire pH range studied (pH 2-7). Fluorescence anisotropy measurements of the intrinsic fluorescence provide evidence suggesting that the pH transition produces a state that does not display greatly increased average flexibility at tryptophan residues. Pepsin digestibility of rhodanese follows the pH dependence of conformational changes reported by activity and physical methods. Rhodanese is resistant to proteolysis above pH 4 but becomes increasingly susceptible as the pH is lowered. The form of the enzyme at pH 3 is cleaved at discrete sites to produce a few large fragments. It appears that pepsin initially cleaves close to one end of the protein and then clips at additional sites to produce species of a size expected for the individual domains into which rhodanese is folded. Overall, it appears that in the pH range between pH 3 and 4, titration of groups on rhodanese leads to opening of the structure to produce a conformation resembling, but more rigid than, the molten globule state that is observed as an intermediate during reversible unfolding of rhodanese.     

Monosaccharide yields and lignin removal from wheat straw in response to catalyst type and pH during mild thermal pretreatment

The influence of various low temperature (140 °C) pretreatments, using different acid and alkaline catalysts and different pH values, was studied for enzymatic hydrolysis of wheat straw. The pretreated wheat straw was treated by a standard blend of Celluclast 1.5L and Novozym 188. While pretreatment at pH 1 gave the highest yield of saccharides in the liquid fraction, the solid fraction was more susceptible to enzymatic attack when pretreated at pH 13. The highest yields were obtained after pretreatment with hydrochloric acid at pH 1, and with sodium hydroxide at pH 13 when enzymatic hydrolysis was employed. A two-step pretreatment strategy at pH 1 (hydrochloric acid) and subsequently at pH 13 (sodium hydroxide) released 69% and 95% of the theoretical maximal amounts of glucose and xylose, respectively. Furthermore, this two-step pretreatment removed 68% of the lignin from the straw with only minor losses of monosaccharides and production of only low amounts of inhibitors. Type of catalyst and pH indeed influenced the monosaccharide yields and lignin removal from wheat straw, and need more attention in the choice of pretreatment strategy.     

Stability and specificity of extracellular protein inhibitor for trypsin from Actinomyces janthinus 118]

Some properties of protein inhibitor for trypsin (TI) from Act. janthinus 118 were studied. It was shown that TI has an antitrypsin activity within a wide pH range with a maximum at about 9,5. At 4 degrees and 20 degrees C TI is stable for 24 hours within the pH range of 6,0--11,0. At 100 degrees C TI is more stable in the slightly acid region of pH than at neutral or alkaline conditions. Trypsin and chymotrypsin inactivate the inhibitor for 8 hours. TI inhibits trypsin, fibrinolysin, subtilisin, pronase and terrilytin, but have no effect on chymotrypsin, thrombin, papain and pepsin. The dissociation constants for the trypsin-inhibitor complex were found to be 1,7.10-8 M, 4,1.10-9 M and 2,4.10-10 M, with casein, p-nitroanilide benzoylarginine and tosylarginine methyl ester used as substrates, respectively. The corresponding dissociation rate constants for the subtilisin-inhibitor complex were equal to 1.10-9 M and 4.10-10 M with casein and carbobenzoxy-L-alanyl-L-alanyl-L-leucin p-nitroanilide used as substrates, respectively.     

4株海生毛壳菌生物学特性的研究

研究了营养、温度、光照和pH值对4株海生毛壳菌(Chaetomium spp.)的生长及孢子形成和萌发的影响,为进一步利用海洋真菌提供理论依据。结果表明:4株毛壳菌在10～35℃均能生长,WHM12、WHM33和WHM34的生长适温为20～30℃,WHM41的生长适温为15～25℃;4株菌在燕麦片琼脂(OA)培养基和YGA培养基上生长最好;在pH值5～10均能生长,但喜中性及偏碱性的环境;全黑暗处理有利于4株菌的生长;除光照外,营养、温度和pH值对其产孢均有影响。     

Evidence and properties of digestive proteases in young mugilidae during the first year of life]

Proteolytic activity was observed in extracts from the digestive tract of Mugil auratus and Mugil capito; its maximum occurs at about pH 2 and 9. Activity in the acid range is mainly found in stomach extracts and the enzyme may be considered as similar to pepsin and cathepsins. In the alkaline range, the main activity is recorded in the region of the pyloric caeca. Activities of the trypsin, chymotrypsin, A and B carboxypeptidases and elastase type are found. The effects of pH and temperature on some activities are studied: optimal pH are about 2.6 and 3,4 with pepsin-like activity, between 8,1 and 8,5 with trypsin-like activity and between 7,8 and 8,5 with chymotrypsin-like activity; incubation optimal temperature (pH 2,2) is found between 35 degrees and 40 degrees.     

Formation of molten globule-like state during acid denaturation of Aspergillus niger glucoamylase

Acid denaturation of Aspergillus niger glucoamylase was studied using different conformational probes. Both far-UV CD spectral signal (MRE_222 nm) and tryptophan fluorescence remained unchanged in the pH range, 7.0–3.0 but decreased significantly below pH 3.0, whereas ANS fluorescence showed a marked increase below pH 1.5. Maximal changes in MRE_222 nm and ANS fluorescence were noticed at pH 1.0. Acid-denatured state of glucoamylase at pH 1.0 retained a significant amount of secondary structure as reflected from far-UV CD spectra but showed a deformed tertiary structure with significant exposure of nonpolar groups as well as tryptophan residues as revealed by increased ANS fluorescence, decreased tryptophan fluorescence and three-dimensional fluorescence spectral signals and increase in K_sv value in acrylamide quenching experiments. Acid-denatured state showed no significant variation in the CD spectral signal throughout the temperature range, 0–100 °C. However, a late cooperative transition was observed upon GdnHCl treatment, compared to the native enzyme. All these results suggested that the acid-denatured state of glucoamylase at pH 1.0 represented the molten globule-like state.     

Spatial structure and antigenic activity of non-glycosylated forms of embryonal prealbumin-1 (EPA-1NG)]

The influence of temperature and pH on the spatial structure of EPA-1NG has been studied by means of circular dichroism and differential UV-spectroscopy, indicating the molecule to consist mainly of beta-structures. A conformational transition in the molecule was observed within the range of 40-50 degrees C. The further temperature elevation (up to 70 degrees C) was accompanied to the complete distortion of the parent conformation, which is reversed after cooling down to 20 degrees C. A correlation of the spectral data with the antigenic activity of genuine EPA-1NG and its carboxymethylated, heat-degraded and pH-denatured derivatives demonstrates that some antigenic determinants of EPA-1NG appear to be topographic.     

Enhanced activity of immobilized pepsin nanoparticles coated on solid substrates compared to free pepsin

In the present work nanoparticles (NPs) of pepsin were generated in an aqueous solution using high-intensity ultrasound, and were subsequently immobilized on low-density polyethylene (PE) films, or on polycarbonate (PC) plates, or on microscope glass slides. The pepsin NPs coated on the solid surfaces have been characterized by HRSEM, TEM, FTIR, XPS and DLS. The amount of enzyme introduced on the substrates, the leaching properties, and the catalytic activity of the immobilized enzyme on the three surfaces are compared. Catalytic activities of pepsin deposited onto the three solid surfaces as well as free pepsin, without sonication, and free pepsin NPs were compared at various pH levels and temperatures using a hemoglobin assay. Compared to native pepsin, pepsin coated onto PE showed the best catalytic activity in all the examined parameters. Pepsin immobilized on glass exhibited better activity than the native enzyme, especially at high temperatures. Enzyme activity of pepsin immobilized on PC was no better than native enzyme activity at all temperatures at pH 2, and only over a narrow pH range at 37 °C was the activity improved over the native enzyme. A remarkable observation is that immobilized pepsin on all the surfaces was still active to some extent even at pH 7, while free pepsin was completely inactive. The kinetic parameters, K_m and V_max were also calculated and compared for all the samples. Relative to the free enzyme, pepsin coated PE showed the greatest improvement in kinetic parameters (K_m = 15 g/L, V_max = 719 U/mg versus K_m = 12.6 g/L and V_max = 787 U/mg, respectively), whereas pepsin coated on PC exhibited the most unfavorable kinetic parameters (K_m = 18 g/L, V_max = 685 U/mg). The values for the anchored enzyme-glass were K_m = 19 g/L, V_max = 763 U/mg.