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1.
The conformational stability of potato cysteine protease inhibitor (PCPI), the second most abundant protease inhibitor group in potato tuber, was investigated at ambient temperature and upon heating using far- and near-UV circular dichroism spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry (DSC). The PCPI isoforms investigated have a highly similar structure at both the secondary and the tertiary level. PCPI isoforms show structural properties similar to those of the potato serine protease inhibitor group and the Kunitz type soybean trypsin inhibitor, a known beta-II protein. Therefore, PCPI isoforms are also classified as members of the beta-II protein subclass. Results show that the thermal unfolding of PCPI isoforms does not follow a two-state mechanism and that at least one intermediate is present. The occurrence of this intermediate is most apparent in the thermal unfolding of PCPI 8.3 as indicated by the presence of two peaks in the DSC thermogram. Additionally, the formation of aggregates (>100 kDa), especially at low scan rates, increases the apparent cooperativity of the unfolding.  相似文献   

2.
The thermal unfolding of potato serine protease inhibitor (PSPI), the most abundant protease inhibitor group in potato tuber, was measured using far UV CD spectroscopy, fluorescence spectroscopy, and DSC. The results indicate that the thermal as well as the guanidinium-induced unfolding of PSPI occurs via a non-two-state mechanism in which at least one stable intermediate is present. Additionally, the occurrence of aggregation, especially at low scan rates, increases the apparent cooperativity of the unfolding and makes the system kinetically rather than thermodynamically controlled. Aggregate formation seems to occur via a specific mechanism of which PSPI in a tetrameric form is the end product and which may involve disulfide interchanges.  相似文献   

3.
The soluble potato proteins are mainly composed of patatin and protease inhibitors. Using DSC and both far-UV and near-UV CD spectroscopy, it was shown that potato proteins unfold between 55 and 75 degrees C. Increasing the ionic strength from 15 to 200 mM generally caused an increase in denaturation temperature. It was concluded that either the dimeric protein patatin unfolds in its monomeric state or its monomers are loosely associated and unfold independently. Thermal unfolding of the protease inhibitors was correlated with a decrease in protease inhibitor activities and resulted in an ionic strength dependent loss of protein solubility. Potato proteins were soluble at neutral and strongly acidic pH values. The tertiary structure of patatin was irreversibly altered by precipitation at pH 5. At mildly acidic pH the overall potato protein solubility was dependent on ionic strength and the presence of unfolded patatin.  相似文献   

4.
The gene of the most abundant protease inhibitor in potato cv. Elkana was isolated and sequenced. The deduced amino acid sequence of this gene showed 98% identity with potato serine protease inhibitor (PSPI), a member of the Kunitz family. Therefore, the most abundant protease inhibitor was considered to be one of the isoforms of PSPI. The PSPI group represents approximately 22% of the total amount of proteins in potato cv. Elkana and is composed of seven different isoforms that slightly differ in isoelectric point. Antibodies were raised against the two most abundant isoforms of PSPI. The binding of these antibodies to PSPI isoforms and protease inhibitors from different groups of protease inhibitor in potato showed that approximately 70% of the protease inhibitors present in potato juice belong to the Kunitz family.  相似文献   

5.
The 7S/11S glycinin equilibrium as found in Lakemond et al. (J. Agric. Food Chem. 2000, 48, xxxx-xxxx) at ambient temperatures influences heat denaturation. It is found that the 7S form of glycinin denatures at a lower temperature than the 11S form, as demonstrated by a combination of calorimetric (DSC) and circular dichroism (CD) experiments. At pH 7.6, at which glycinin is mainly present in the 11S form, the disulfide bridge linking the acidic and the basic polypeptides is broken during heat denaturation. At pH 3.8, at which glycinin has dissociated partly into the 7S form, and at pH 5.2 this disruption does not take place, as demonstrated by solubility and gel electrophoretic experiments. A larger exposure of the acidic polypeptides (Lakemond et al., 2000) possibly correlates with a higher endothermic transition temperature and with the appearance of an exothermic transition as observed with DSC. Denaturation/aggregation (studied by DSC) and changes in secondary structure (studied by far-UV CD) take place simultaneously. Generally, changes in tertiary structure (studied by near-UV CD) occur at lower temperatures than changes in secondary structure.  相似文献   

6.
In the present study the structural properties of potato protease inhibitor 1 (PI-1) were studied as a function of temperature to elucidate its precipitation mechanism upon heating. A cDNA coding for PI-1 from cv. Bintje was cloned and expressed in Pichia pastoris. Using the recombinant PI-1 it was suggested that PI-1 behaves as a hexameric protein rather than as a pentamer, as previously proposed in the literature. The recombinant protein seems either to have a predominantly unordered structure or to belong to the beta-II proteins. Differential scanning calorimetry analysis of PI-1 revealed that its thermal unfolding occurs via one endothermic transition in which the hexameric PI-1 probably unfolds, having a dimer instead of a monomer as cooperative unit. The transition temperature for the recombinant PI-1 was 88 degrees C. Similar results were obtained for a partially purified pool of native PI-1 from cv. Bintje.  相似文献   

7.
Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated in phosphate buffer at temperatures between 40 and 94 degrees C for 10 min, cooled, and analyzed using near-UV and far-UV circular dichroism (CD). The decrease in near-UV CD intensity at 293 nm (Deltaepsilon(293)) could be analyzed in terms of a two-state model, and the stability was beta-Lg C > beta-Lg A > beta-Lg B on the basis of the midpoint temperatures for samples heated at pH 6.7 and 7.4. However, the slopes of the curves at the midpoint temperature for variant A were generally less than those for beta-Lg B and beta-Lg C, indicating that the substitution of Val (beta-Lg A) for Ala (beta-Lg B or beta-Lg C) at position 118 had altered the entropic contribution to unfolding of the protein. The changes in CD at 270 nm (Deltaepsilon(270)), an index of significant alteration to disulfide bond dihedral angles, occurred at higher temperatures than those for the Deltaepsilon(293) results. The far-UV CD showed some small changes as a consequence of heat treatment, and the shifts at 205 nm ([theta](205)) fitted a two-state model. Plotting the changes in both Deltaepsilon(293) and [theta](205) against the loss of nativelike and sodium dodecyl sulfate-monomeric protein (assessed by polyacrylamide gel electrophoresis) showed a strong 1:1 relationship between Deltaepsilon(293) or [theta](205) and the loss of nativelike beta-Lg. These results indicated that the initial irreversible stage in the heat-induced aggregation of beta-Lg (nativelike monomer to unfolded monomer) altered the chirality of the environment of Trp(19) and modified the secondary structure of beta-Lg slightly. The differences in the behavior of variants A-C were explicable on the basis of generalized electrostatic and hydrophobicity effects as well as specific amino acid effects.  相似文献   

8.
The mechanism that leads to a decreased aggregation of beta-lactoglobulin in the presence of dextran sulfate and lambda-carrageenan was investigated by assessing changes in the denaturation thermodynamics and protein structure. Differential scanning calorimetry results showed that the denaturation temperature (Tp) was about 4.6 degrees C higher in the presence of dextran sulfate, as compared with beta-lactoglobulin alone, whereas in the presence of lambda-carrageenan the difference in Tp was about 1.2 degrees C. Changes in protein structure studies using near-UV circular dichroism (CD) provided support for the calorimetric results. The transition midpoint (Tm) for denaturation of beta-lactoglobulin was about 5 degrees C higher in the presence of dextran sulfate than that found with beta-lactoglobulin alone and about 2 degrees C in the presence of lambda-carrageenan. Thermal modifications of the tertiary structure of beta-lactoglobulin were irreversible at temperatures above 67 degrees C; the addition of dextran sulfate reduced the extent of such modifications. Far-UV CD studies indicated that the addition of dextran sulfate or lambda-carrageenan did not affect secondary structure changes of beta-lactoglobulin upon heating. These studies indicate that dextran sulfate and lambda-carrageenan can enhance the stability of beta-lactoglobulin and thereby inhibit heat denaturation and aggregation.  相似文献   

9.
The secondary structure of Kluyveromyces lactis beta-galactosidase was determined by circular dichroism. It is mainly a beta-type protein, having 22% beta-turns, 14% parallel beta-sheet, 25% antiparallel beta-sheet, 34% unordered structure, and only 5% alpha-helix. The structure-activity relationship as a function of the pH was also studied. The pH conditions leading to the highest secondary structure content (100% ellipticity) of the enzyme was found at pH 7.0; at pH 6.5-7.0, the percent ellipticity decreased slightly, suggesting little structural change, but the activity decreased significantly, probably because of variations in critical residues. On the other hand, at pH's above 7.0, a more noticeable change in ellipticity was observed due to structural changes; the CD analysis showed a small increase in the helical content toward higher pH, whereas the maximum activity was found at pH 7.5, meaning that the changes produced in the secondary structure at this pH favored the interaction between the enzyme and the substrate.  相似文献   

10.
Two protease inhibitors of 67 and 18 kDa, respectively, were purified from glassfish, Liparis tanakai, eggs by affinity chromatography. The smaller protein was purified with a yield and purity of 0.25% and 49.69-fold, respectively, and was characterized for further study. The glassfish egg protease inhibitor exhibited stability between 50 and 65 degrees C in an alkaline environment (pH 8). It was shown to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 4.44 nM. Potent glassfish protease inhibitor with N-Val-Gly Ser-Met-Thr-Gly-Gly-Phe-Thr-Asp-C amino acid residues was synthesized and its inhibitory activity was compared. Moreover, the 18-kDa protein inhibited cathepsin, a cysteine protease, more effectively than did egg white protease inhibitor, whereas the reverse was true for papain. Glassfish egg protease inhibitor is classified as a member of the family I cystatins.  相似文献   

11.
Lunasin and Bowman-Birk protease inhibitor (BBI) are two soybean peptides to which health-promoting properties have been attributed. Concentrations of these peptides were determined in skim fractions produced by enzyme-assisted aqueous extraction processing (EAEP) of extruded full-fat soybean flakes (an alternative to extracting oil from soybeans with hexane) and compared with similar extracts from hexane-defatted soybean meal. Oil and protein were extracted by using countercurrent two-stage EAEP of soybeans at 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h. Protein-rich skim fractions were produced from extruded full-fat soybean flakes using different enzyme strategies in EAEP: 0.5% protease (wt/g extruded flakes) used in both extraction stages; 0.5% protease used only in the second extraction stage; no enzyme used in either extraction stage. Countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes was used as a control. Protein extraction yields increased from 66% to 89-96% when using countercurrent two-stage EAEP with extruded full-fat flakes compared to 85% when using countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes. Extruding full-fat soybean flakes reduced BBI activity. Enzymatic hydrolysis reduced BBI contents of EAEP skims. Lunasin, however, was more resistant to both enzymatic hydrolysis and heat denaturation. Although using enzymes in both EAEP extraction stages yielded the highest protein and oil extractions, reducing enzyme use to only the second stage preserved much of the BBI and Lunasin.  相似文献   

12.
Bovine immunoglobulin G (IgG) solutions were subjected to pulsed electric fields (PEF) or heat treatment to investigate the effect of processing on secondary structure monitored using circular dichroism spectrometry. Under heat treatment, the critical temperature for bovine IgG to change secondary structure at neutral pH in borate buffer is 72 degrees C. A conversion of the secondary structure from beta-sheets into random coils along with the loss of immunoactivity of bovine IgG was observed when heated at 82 degrees C for 120 s. In contrast, PEF treatment at 41.1 kV/cm for 54 mus with bipolar pulses (outlet at 43.8 degrees C) caused no detectable changes in the secondary structure or the thermal stability of secondary structure. A shape factor, S (200nm) over (217nm), ratio of magnitude of the positive CD band at 200 nm to that of the negative CD band at 217 nm, was closely correlated to the immunoactivity of bovine IgG (r(2) = 0.99) and quantifies changes of secondary structure.  相似文献   

13.
The secondary structure of protein adsorbed at the emulsion interface has been studied in refractive index matched emulsions using the techniques of circular dichroism (CD) and Fourier transform infrared spectroscopy. Bovine serum albumin (BSA) and bovine beta-lactoglobulin (betalg) stabilized emulsions were studied, and the refractive index was altered by the addition of glycerol or polyethylene glycol. The effect of additive on the solution and adsorbed protein structure in addition to the effect of adsorption time was considered. Both adsorption and glycerol addition alter protein secondary structure; however, the majority of secondary structure remains. Small changes are observed in the secondary structure of adsorbed protein with time. Near-ultraviolet CD studies showed the effect of glycerol and adsorption on the aromatic groups. BSA showed small changes both upon the addition of glycerol to protein in solution and upon adsorption. betalg showed slightly larger changes upon the addition of glycerol to protein in solution and a larger change upon adsorption.  相似文献   

14.
High-pressure processing (HPP) was utilized to induce unfolding of beta-lactoglobulin (beta-LG). beta-Lactoglobulin solutions at concentrations of 0.5 mg/mL, in pH 7.5 phosphate buffer, were pressure treated at 510 MPa for 10 min at either 8 or 24 degrees C. The secondary structure, as determined by circular dichroism (CD), of beta-LG processed at 8 degrees C appeared to be unchanged, whereas beta-LG processed at 24 degrees C lost alpha-helix structure. Tertiary structures for beta-LG, as determined by near-UV CD, intrinsic protein fluorescence spectroscopy, hydrophobic fluorescent probe binding, and thiol group reactivity, were changed following processing at either temperature. The largest changes to tertiary structure were observed for the samples processed at 24 degrees C. Model solutions containing the pressure-treated beta-LG showed significant decreases in surface tension at liquid-air interfaces with values of 54.00 and 51.69 mN/m for the samples treated at 24 and 8 degrees C, respectively. In comparison, the surface tension for model solutions containing the untreated control was 60.60 mN/m. Changes in protein structure during frozen and freeze-dried storage were also monitored, and some renaturation was observed for both storage conditions. Significantly, the sample pressure-treated at 8 degrees C continued to display the lowest surface tension.  相似文献   

15.
The body complex of the soybean seed (BCSS) was isolated from the single cells (27.2%) by a sequential procedure of autoclaving with water, cellulase digestion for the primary cell wall, pectinase digestion for the secondary cell wall, and defatting with hexane washing. Its characteristics were then investigated. The defatted BCSS (DBCSS) consisted of protein (76.5%) and mannose-rich carbohydrates (3.2%). Screening of the food-processing protease for the digestion of DBCSS was carried out, and a kind of alkaline protease was selected. The inner protein of DBCSS was easily extracted with 0.1 M sodium carbonate buffer, pH 10, and the insoluble shell of the body complex (SDBCSS) was left. SDBCSS consisted of hydrophobic amino acid-rich protein. SDBCSS was easily digested by the selected alkaline protease. SDBCSS was dissolved by boiling with sodium dodecyl sulfate-mercaptoethanol, and it was found to consist of a protein of approximately 3 kDa. The high enzymatic digestion including the selected protease for soybean seed and defatted soybean meal was carried out; both were extracted and digested with a yield of >99.5%. The final indigestible residue was found as paired hexagonal and filamentous organs of the soybean cells.  相似文献   

16.
Purification of a lipoxygenase enzyme from the cultivar Tresor of durum wheat semolina (Triticum turgidum var. durum Desf) was reinvestigated furnishing a new procedure. The 895-fold purified homogeneous enzyme showed a monomeric structure with a molecular mass of 95 +/- 5 kDa. Among the substrates tested, linoleic acid showed the highest k(cat)/K(m) value; a beta-carotene bleaching activity was also detected. The enzyme optimal activity was at pH 6. 8 on linoleic acid as substrate and at pH 5.2 for the bleaching activity on beta-carotene, both assayed at 25 degrees C. The dependence of lipoxygenase activity on temperature showed a maximum at 40 degrees C for linoleic acid and at 60 degrees C for bleaching activity on beta-carotene. The amino acid composition showed the presence of only one tryptophan residue per monomer. Far-UV circular dichroism studies carried out at 25 degrees C in acidic, neutral, and basic regions revealed that the protein possesses a secondary structure content with a high percentage of alpha- and beta-structures. Near-UV circular dichroism, at 25 degrees C and at the same pH values, pointed out a strong perturbation of the tertiary structure in the acidic and basic regions compared to the neutral pH condition. Moreover, far-UV CD spectra studying the effects of the temperature on alpha-helix content revealed that the melting point of the alpha-helix is at 60 degrees C at pH 5.0, whereas it was at 50 degrees C at pH 6.8 and 9.0. The NH(2)-terminal sequence allowed a homology comparison with other lipoxygenase sequences from mammalian and vegetable sources.  相似文献   

17.
Protease inhibitors from potato juice of cv. Elkana were purified and quantified. The protease inhibitors represent ca. 50% of the total soluble proteins in potato juice. The protease inhibitors were classified into seven different families: potato inhibitor I (PI-1), potato inhibitor II (PI-2), potato cysteine protease inhibitor (PCPI), potato aspartate protease inhibitor (PAPI), potato Kunitz-type protease inhibitor (PKPI), potato carboxypeptidase inhibitor (PCI), and "other serine protease inhibitors". The most abundant families were the PI-2 and PCPI families, representing 22 and 12% of all proteins in potato juice, respectively. Potato protease inhibitors show a broad spectrum of enzyme inhibition. All the families (except PCI) inhibited trypsin and/or chymotrypsin. PI-2 isoforms exhibit 82 and 50% of the total trypsin and chymotrypsin inhibiting activity, respectively. A strong variation within the latter activities was shown within one family and between protease inhibitor families.  相似文献   

18.
The interface of 10 or 25% (w/v) bovine serum albumin (BSA), pH 7, buffered solution against mineral or corn oil was studied with a Raman microscope. A gradient of distribution of protein and oil at the interface was observed. The difference spectrum obtained by subtracting the spectrum of mineral or corn oil from that of the BSA/oil interface indicated interactions involving different functional groups of the BSA and the oil molecules. Against mineral oil, the BSA spectrum showed reduced intensity of the tryptophan band at 750 cm(-1) and reduced intensity ratio of the tyrosine doublet at 850-830 cm(-1), indicating changes in the microenvironment of these hydrophobic residues. A negative band at 2850 cm(-1) indicated the involvement of the CH groups in the mineral oil. However, the amide regions, normally assigned to protein secondary structure, were not significantly changed. When the spectrum of BSA was subtracted from the BSA/mineral oil interface spectrum, the resultant difference spectrum showed changes of symmetric and antisymmetric CCC stretches at 980 and 1071 cm(-1), respectively. In contrast, the difference spectrum of BSA/corn oil interface - BSA showed a decrease of CH(2) symmetric stretching at 2850 cm(-1) and a decrease of unsaturated fatty acid hydrocarbon chain stretch at 3010 cm(-1). Raman spectroscopy is a useful tool to study the nature of protein-lipid interactions.  相似文献   

19.
In the present study emulsions were made with various potato protein preparations, which varied in protease inhibitor and patatin content. These emulsions were characterized with respect to average droplet size, plateau surface excess, and the occurrence of droplet aggregation. Droplet aggregation occurred only with potato protein preparations that contained a substantial amount of protease inhibitors and could be prevented only at pH 3. The average droplet size of the emulsions made with potato proteins appeared to be related to the patatin content of the preparation used. Average droplet size was found to be dominated by the patatin-catalyzed lipolytic release of surface active fatty acids and monoglycerides from the tricaprylin oil phase during the emulsification process. Addition of monoglycerides and especially fatty acids, at concentrations representative of those during emulsification, was shown to cause a stronger and much faster decrease of the interfacial tension than that with protein alone and to result in a drastic decrease in droplet size. The patatin used was shown to have a lipolytic activity of 820 units/g with emulsified tricaprylin as the substrate. Because of the droplet aggregating properties of the protease inhibitors, the patatin-rich potato preparations seem to be the most promising for food emulsion applications over a broad pH range, provided the lipolytic activity can be diminished or circumvented.  相似文献   

20.
Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) were used to study changes in the conformation of globulin from common buckwheat (Fagopyrum esculentum Moench) (BWG) under various environmental conditions. The IR spectrum of the native BWG showed several major bands from 1691 to 1636 cm(-1) in the amide I' region, and the secondary structure composition was estimated as 34.5% beta-sheets, 20.0% beta-turns, 16.0% alpha-helices, and 14.4% random coils. Highly acidic and alkaline pH conditions induced decreases in beta-sheet and alpha-helical contents, as well as in denaturation temperature (Td) and enthalpy of denaturation (DeltaH), as shown in the DSC thermograms. Addition of chaotropic salts (1.0 M) caused progressive decreases in ordered structures and thermal stability following the lyotropic series of anions. The presence of several protein structure perturbants also led to changes in IR band intensities and DSC thermal stabilities, suggesting protein unfolding. Intermolecular antiparallel beta-sheet (1620 and 1681 cm(-1)) band intensities started to increase when BWG was heated to 90 degrees C, suggesting the initiation of protein aggregation. Increasing the time of the preheat treatment (at 100 degrees C) caused progressive increases in Td and pronounced decreases in DeltaH, suggesting partial denaturation and reassociation of protein molecules.  相似文献   

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