Guanidine Hydrochloride Denaturation of Glycosylated and Deglycosylated Stem Bromelain

Glycosylation is one of the major naturally occurring covalent modifications of proteins. We have used stem bromelain, a thiol protease with a single, N-glycosylated polypeptide chain as a model to investigate the role of glycosylation of proteins. Periodate oxidation was used to obtain the deglycosylated form of the enzyme. Denaturation studies in the presence of guanidine hydrochloride (Gn·HCl) were performed using fluorescence and circular dichroism spectroscopy. The glycosylated stem bromelain was found to be stabilized by 1.9 kcal/mol as compared to the deglycosylated one. At a given concentration of denaturant, the fraction of denatured protein was higher in the case of deglycosylated stem bromelain. In short, deglycosylated bromelain showed more susceptibility towards guanidine hydrochloride denaturation, indicating the contribution of the carbohydrate part of the glycoprotein to the stability of the enzyme.     

Low versus high molecular weight poly(ethylene glycol)-induced states of stem bromelain at low pH: stabilization of molten globule and unfolded states

The effect of low, medium, and high molecular weight poly(ethylene glycol) (e.g., PEG-400, -6000, and -20,000) on the structure of the acid unfolded state of unmodified stem bromelain (SB) obtained at pH 2.0 has been studied by various spectroscopic methods. The conformation of stem bromelain at pH 2.0 exhibits substantial loss of secondary structure and almost complete loss of native tertiary contacts and has been termed the acid unfolded state (A(U)). Addition of PEG-400 to A(U) led to an increase in the mean residue ellipticity (MRE) value at 222 nm, indicating formation of alpha-helical structure. On the other hand, PEG-6000 and 20,000 led to a decrease in the MRE value at 222 nm, indicating unfolding of the A(U) state. Interestingly, at 70% (w/v) PEG-400 and 40% (w/v) PEG-20,000, MRE values at 222 nm almost approach the native state at pH 7.0 and the unfolded state (6 M GnHCl) of stem bromelain, respectively. The probes for tertiary structure showed formation of nonnative tertiary contacts in the presence of 70% (w/v) PEG-400, while 40% (w/v) PEG-6000 and 20,000 were found to stabilize the unfolded state of SB. An increase in binding of 1-anilino 8-naphthalene sulfonic acid and a decrease in fractional accessibility of tryptophan residues (f(a)) compared to A(U) in the presence of 70% PEG-400 indicate that the PEG-400-induced state has a significant amount of exposed hydrophobic patches and is more compact than A(U). The results imply that the PEG-400-induced state has characteristics of molten globule, and higher molecular weight PEGs led to the unfolding of the A(U) state.     

菠萝果蛋白酶生产工艺的改进

本试验研究了菠萝果蛋白酶生产工艺中的各个重要环节。结果表明:用0.08～0.1％的丹宁作沉淀剂较适宜,既可保持产品质量,又可兼顾产品产量;酶膏在-12℃以下低温冻结和真空干燥两个因素能显著提高产品酶活性;1000 ug/g硫代硫酸钠加62.5 ug/g半胱氨酸是菠萝果蛋白酶活性的有效保护剂,可使酶活性比对照提高32.57％;提取过程中用抗坏血酸、乙二胺四乙酸二钠、氯化钠和醋酸锌溶液对酶复合物进行洗涤可有效地提高产品的活性和质量。     

Effect of phosphorylation on the thermal and light stability of the thylakoid membranes

Higher plant thylakoid membranes contain a protein kinase that phosphorylates certain threonine residues of light-harvesting complex II (LHCII), the main light-harvesting antenna complexes of photosystem II (PSII) and some other phosphoproteins (Allen, Biochim Biophys Acta 1098:275, 1992). While it has been established that phosphorylation induces a conformational change of LHCII and also brings about changes in the lateral organization of the thylakoid membrane, it is not clear how phosphorylation affects the dynamic architecture of the thylakoid membranes. In order to contribute to the elucidation of this complex question, we have investigated the effect of duroquinol-induced phosphorylation on the membrane ultrastructure and the thermal and light stability of the chiral macrodomains and of the trimeric organization of LHCII. As shown by small angle neutron scattering on thylakoid membranes, duroquinol treatment induced a moderate (~10%) increase in the repeat distance of stroma membranes, and phosphorylation caused an additional loss of the scattering intensity, which is probably associated with the partial unstacking of the granum membranes. Circular dichroism (CD) measurements also revealed only minor changes in the chiral macro-organization of the complexes and in the oligomerization state of LHCII. However, temperature dependences of characteristic CD bands showed that phosphorylation significantly decreased the thermal stability of the chiral macrodomains in phosphorylated compared to the non-phosphorylated samples (in leaves and isolated thylakoid membranes, from 48.3°C to 42.6°C and from 47.5°C to 44.3°C, respectively). As shown by non-denaturing PAGE of thylakoid membranes and CD spectroscopy on EDTA washed membranes, phosphorylation decreased by about 5°C, the trimer-to-monomer transition temperature of LHCII. It also enhanced the light-induced disassembly of the chiral macrodomains and the monomerization of the LHCII trimers at 25°C. These data strongly suggest that phosphorylation of the membranes considerably facilitates the heat- and light-inducible reorganizations in the thylakoid membranes and thus enhances the structural flexibility of the membrane architecture.     

Application of an aqueous two‐phase micellar system to extract bromelain from pineapple (Ananas comosus) peel waste and analysis of bromelain stability in cosmetic formulations

Bromelain is a set of proteolytic enzymes found in pineapple (Ananas comosus) tissues such as stem, fruit and leaves. Because of its proteolytic activity, bromelain has potential applications in the cosmetic, pharmaceutical, and food industries. The present study focused on the recovery of bromelain from pineapple peel by liquid–liquid extraction in aqueous two‐phase micellar systems (ATPMS), using Triton X‐114 (TX‐114) and McIlvaine buffer, in the absence and presence of electrolytes CaCl₂ and KI; the cloud points of the generated extraction systems were studied by plotting binodal curves. Based on the cloud points, three temperatures were selected for extraction: 30, 33, and 36°C for systems in the absence of salts; 40, 43, and 46°C in the presence of KI; 24, 27, and 30°C in the presence of CaCl₂. Total protein and enzymatic activities were analyzed to monitor bromelain. Employing the ATPMS chosen for extraction (0.5 M KI with 3% TX‐114, at pH 6.0, at 40°C), the bromelain extract stability was assessed after incorporation into three cosmetic bases: an anhydrous gel, a cream, and a cream‐gel formulation. The cream‐gel formulation presented as the most appropriate base to convey bromelain, and its optimal storage conditions were found to be 4.0 ± 0.5°C. The selected ATPMS enabled the extraction of a biomolecule with high added value from waste lined‐up in a cosmetic formulation, allowing for exploration of further cosmetic potential. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:937–945, 2015     

Effect of pH and denaturants on the folding and stability of murine interleukin-6.

The conformation and stability of a recombinant mouse interleukin-6 (mIL-6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea-gradient gel electrophoresis, and near- and far-ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL-6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL-6, as judged by both urea-gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0-10.0. Sedimentation equilibrium experiments indicated that mIL-6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp-36 and Trp-160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17-residue synthetic peptide, peptide H1, which corresponds to an N-terminal region of mIL-6 (residues Val-27-Lys-43). The pH-dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of alpha-helix-promoting solvents. Replacement of His-33 with Ala-33 in peptide H1 alleviated a significant portion of the pH-dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His-33 with the indole ring of Trp-36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL-6.     

Effect of invertase on the batch foam fractionation of bromelain

Foam fractionation can be used to enrich a hydrophobic protein such as bromelain from an aerated dilute protein solution because the protein foams. On the other hand, a protein such as invertase, which is hydrophilic, is not likely to foam under similar aerated conditions. While a foam fractionation process may not be approapriate for recovering a hydrophilic protein alone, it is of interest to see how that non-foaming protein affects the foaming protein when the two are together in a mixture. The bromelain enrichment, activity and mass recovery were observed as a function of the solution pH in order to explore how invertase can affect the recovery of bromelain in a foam fractionation process.     

Oriented immobilization of stem bromelain via the lone histidine on a metal affinity support

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from the stem of the pineapple plant (Ananas comosus) and is unique for it contains a single histidine residue (His-158) in the polypeptide. Based on the technology of protein separation with immobilized metal ion affinity chromatography (IMAC), a method for oriented immobilization of bromelain was selected. Bromelain was successfully immobilized on iminodiacetic acid carrier Sepharose 6B. Cu²⁺ complexed with iminodiacetate (IDA) was used as the chelating ligand to bind the lone histidine on bromelain. Simultaneously, preparation of a high affinity immobilized preparation was attempted using a soluble cross-linked preparation of bromelain on Cu-IDA-Sepharose. However this second method proved unsuccessful, possibly due to poor histidine accessibility in the cross-linked preparation. The immobilized preparation obtained using uncrosslinked bromelain was more resistant to thermal inactivation, as evidenced by retention of over enzyme 50% activity after incubation at 60 °C, as compared to 20% retained by the native enzyme. The immobilized preparation also exhibited a broader pH-activity profile in acidic range. The native, immobilized and soluble cross-linked bromelain showed apparent Michaelis constant (K_m) values of 1.08, 0.42, 1.56 mg/ml, respectively, using casein as the substrate. While the maximum velocity (V_max) values of the soluble and immobilized preparations were comparable, cross-linked preparation showed a 20% decrease, suggesting inactivation. The mild conditions used for predominantly oriented immobilization exploiting the unique property of single histidine, the high recovery of immobilized preparations, the stability, reusability and the regenerability of the matrix are the main features of the method reported here.     

pH effects on the stability and dimerization of procaspase-3

pH-dependent conformational changes are known to occur in dimeric procaspase-3, and they have been shown to affect the rate of automaturation. We studied the equilibrium unfolding of procaspase-3(C163S) as a function of pH (between pH 8.5 and pH 4) in order to examine these changes in the context of folding and stability. The data show that the procaspase dimer undergoes a pH-dependent dissociation below pH 5, so that the protein is mostly monomeric at pH 4. Consistent with this, the dimer unfolds via a four-state process between pH 8.5 and pH 4.75, in which the native dimer isomerizes to a dimeric intermediate, and the dimeric intermediate dissociates to a monomer, which then unfolds. In contrast, a small protein concentration dependence was observed by circular dichroism, but not by fluorescence emission, at pH 4.5 and pH 4.2. There was no protein-concentration dependence to the data collected at pH 4. Overall, the results are consistent with the redistribution of the population of native dimer (N(2)) to dimeric intermediate (I(2)) to monomeric intermediate (I), as the pH is lowered so that at pH 4, the "native" ensemble resembles the monomeric intermediate (I) observed during unfolding at higher pH. An emerging picture of the monomeric procaspase is discussed. Procaspase-3 is most stable at pH approximately 7 (24-26 kcal/mol), and while the stability decreased with pH, it was observed that dimerization contributes the majority (>70%) of the conformational free energy.     

Influence of salts and alcohols on the conformation of partially folded intermediate of stem bromelain at low pH

The effect of salts and alcohols was examined on the partially folded intermediate (PFI) state of stem bromelain reported at low pH (Haq, Rasheedi, and Khan (2002) European Journal of Biochemistry 269, 47-52) by a combination of optical methods like circular dichroism, intrinsic fluorescence and ANS binding. ESI mass spectrometry was also performed to see the effect, if any, on the overall tertiary structure of the protein. Increase in ionic strength by the addition of salts resulted in folded structures somewhat different from the native enzyme. Salt-induced intermediates are characterized by increase in helical content and a significantly reduced exposure of hydrophobic clusters relative to the state at pH 2.0. The emission wavelength maximum of intrinsic fluorescence was shifted towards that of native enzyme. ESI-MS data show decreased accessibility of ionizable/protonation sites suggestive of a folded structure. On the other hand, alcohol-induced intermediates though exhibiting increased helical content are apparently largely unfolded as observed by ESI. Thermal denaturation of a representative intermediate, each from the group of salts and alcohols examined, was also performed to check their relative stabilities. While the alcohol-induced state showed a cooperative thermal transition, the salt-induced state shows non-cooperative thermal denaturation.     

The biocatalytic activity of bromelain in ester synthesis reaction: difference between the intrinsic lipase activity and thermal catalysis

Biocatalytic activities of bromelain preparations were carried out in proteolytic (4500 units g^–1), lipolytic (67 units g^–1) and, more particularly, in fatty acid ester synthetic reactions. The ester synthesis reactions were studied and several thermodynamic parameters and non-biological reference reactions were also investigated. Only temperature had a strong influence on the maximum reaction yield (30% after 10 days) and revealed that thermal catalysis, which exists in esterification, raises doubts concerning the real biocatalytic activity of the plant extract. When this thermal catalysis is taken into account, the intrinsic lipase activity of the bromelain preparations in esterification reactions is nil.     

Effects of calcium binding on the structure and stability of human growth hormone

Thermodynamic analysis of calcium ions binding to human growth hormone (hGH) was done at 27 °C in NaCl solution, 50 mM, using different techniques. The binding isotherm for hGH-Ca²⁺ was obtained by two techniques of ionmetry, using a Ca²⁺-selective membrane electrode, and isothermal titration calorimetry. Results obtained by two ionmetric and calorimetric methods are in good agreement. There is a set of three identical and non-interacting binding sites for calcium ions. The intrinsic dissociation equilibrium constant and the molar enthalpy of binding are 52 μM and −17.4 kJ/mol, respectively. Temperature scanning UV–vis spectroscopy was applied to elucidate the effect of Ca²⁺ binding on the protein stability, and circular dichroism (CD) spectroscopy was used to show the structural change of hGH due to the metal ion interaction. Calcium ions binding increase the protein thermal stability by increasing of the alpha helix content as well as decreasing of both beta and random coil structures.     

Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl),pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65°C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasingpH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55°C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5°C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.     

Monitoring the effects of strong cosolvent hexafluoroisopropanol in investigation of the tetrameric structure and stability of K-channel KcsA

Adsorption of small chain alcohols into lipid membranes significantly changes the conformational states of intrinsic membrane proteins. In this study, the effects of membrane-active strong cosolvent hexafluoroisopropanol (HFIP) on the intrinsic tetrameric stability of potassium channel KcsA were investigated. Presence of acidic phosphatidylglycerol (PG) in non-bilayer phosphatidylethanolamine (PE) or bilayer phosphatidylcholine (PC) significantly increased the tetrameric stability compared to zwitterionic pure PC bilayers. The stabilizing effect of PG in both lipid bilayers was completely abolished upon deletion of the membrane-anchored N-terminus. Tryptophan fluorescence and circular dichroism experiments indicated that HFIP destabilizes the tetramer possibly via drastic changes in the lateral pressure profile close to the membrane-water interface. The data suggest that HFIP disturbs the ionic, H-bonding and hydrophobic interactions among KcsA subunits where N-terminus presumably plays a crucial role in determining the channel proper folding and tetrameric structure via ionic/H-bond interactions between the helix dipole and the membrane lipids.     

The effects of time,temperature, and pH on the stability of PDU bacterial microcompartments

Bacterial microcompartments (MCPs) are subcellular organelles that are composed of a protein shell and encapsulated metabolic enzymes. It has been suggested that MCPs can be engineered to encapsulate protein cargo for use as in vivo nanobioreactors or carriers for drug delivery. Understanding the stability of the MCP shell is critical for such applications. Here, we investigate the integrity of the propanediol utilization (Pdu) MCP shell of Salmonella enterica over time, in buffers with various pH, and at elevated temperatures. The results show that MCPs are remarkably stable. When stored at 4°C or at room temperature, Pdu MCPs retain their structure for several days, both in vivo and in vitro. Furthermore, Pdu MCPs can tolerate temperatures up to 60°C without apparent structural degradation. MCPs are, however, sensitive to pH and require conditions between pH 6 and pH 10. In nonoptimal conditions, MCPs form aggregates. However, within the aggregated protein mass, MCPs often retain their polyhedral outlines. These results show that MCPs are highly robust, making them suitable for a wide range of applications.     

Effect of Heat and pH Denaturation on the Structure and Conformation of Recombinant Human Hepatic Stimulator Substance

Hepatic stimulator substance (HSS) is a novel liver-specific growth-promoting factor. Although HSS has been successfully crystallized, several properties of this protein have yet to be determined. This study shows that recombinant human HSS (rhHSS) is a dimer with a molecular mass of 31 kDa, The protein is weakly acidic and has an isoelectric point (pI) of 4.50. rhHSS was able to protect hepatoma cells from H₂O₂-induced apoptosis and to stimulate cell growth. The recombinant protein was thermostable up to 80°C and resistant to changes in pH, as determined by synchronous fluorescence and far-UV circular dichroism (CD). Within the range of pH 4.0–10.0, rhHSS assumed a folded conformation identical to the secondary structure of the original, native protein and a native-like far-UV CD spectrum. Denatured rhHSS could be partly reconstituted with respect to its structure, but not its activity. Thus, rhHSS is a structurally stable protein insensitive to thermal and acid–alkaline denaturation.     

A meta-analysis of the activity,stability, and mutational characteristics of temperature-adapted enzymes

Understanding the characteristics that define temperature-adapted enzymes has been a major goal of extremophile enzymology in recent decades. In the present study, we explore these characteristics by comparing psychrophilic, mesophilic, and thermophilic enzymes. Through a meta-analysis of existing data, we show that psychrophilic enzymes exhibit a significantly larger gap (T_g) between their optimum and melting temperatures compared with mesophilic and thermophilic enzymes. These results suggest that T_g may be a useful indicator as to whether an enzyme is psychrophilic or not and that models of psychrophilic enzyme catalysis need to account for this gap. Additionally, by using predictive protein stability software, HoTMuSiC and PoPMuSiC, we show that the deleterious nature of amino acid substitutions to protein stability increases from psychrophiles to thermophiles. How this ultimately affects the mutational tolerance and evolutionary rate of temperature adapted organisms is currently unknown.     

Influence of phosphate anions on the stability of immobilized enzymes. Effect of enzyme nature,immobilization protocol and inactivation conditions

A destabilizing effect at pH 7 of sodium phosphate on several lipases immobilized via interfacial activation is shown in this work. This paper investigates if this destabilizing effect is extended to other inactivation conditions, immobilization protocols or even other immobilized enzymes (ficin, trypsin, β-galactosidase, β-glucosidase, laccase, glucose oxidase and catalase). As lipases, those from Candida antarctica (A and B), Candida rugosa and Rhizomucor miehei have been used. Results confirm the very negative effect of 100 mM sodium phosphate at pH 7.0 for the stability of all studied lipases immobilized on octyl agarose, while using glutaraldehyde-support the effect is smaller (still very significant using CALA) and in some cases the effect disappeared (e.g., using CALB). The change of the pH to 5.0 or 9.0, or the addition of 1 M NaCl reduced the negative effect of the phosphate in some instances (e.g., at pH 5.0, this negative effect is only relevant for CALB). Regarding the other enzymes, only the monomeric β-galactosidase from Aspergillus oryzae is strongly destabilized by the phosphate buffer. This way, the immobilization protocol and the inactivation conditions strongly modulate the negative effect of sodium phosphate on the stability of immobilized lipases, and this effect is not extended to other enzymes.     

Human Calprotectin： Effect of Calcium and Zinc on its Secondary and Tertiary Structures, and Role of pH in its Thermal Stability

Calprotectin, a heterodimeric complex belonging to the S 100 protein family, has been found predominantly in the cytosolic fraction of neutrophils. In the present study, human calprotectin was purified from neutrophils using two-step ion exchange chromatography. The purified protein was used for circular dichroism study and fluorescence analysis in the presence of calcium and zinc at physiological concentrations, as well as for assessment of its inhibitory activity on the K562 leukemia cell line. The thermal stability of the protein at pH 7.0 （physiological pH） and 8.0 （similar to intestinal pH） was also compared. The results of cell proliferation analysis revealed that human calprotectin initiated growth inhibition of the tumor cells in a dose- dependent manner. The intrinsic fluorescence emission spectra of human calprotectin （50 ktg/ml） in the presence of calcium and zinc ions show a reduction in fluorescence intensity, reflecting a conformational change within the protein with exposure of aromatic residues to the protein surface that is important for the biological function of calprotectin. The far ultraviolet-circular dichroism spectra of human calprotectin in the presence of calcium and zinc ions at physiological concentrations show a decrease in the m-helical content of the protein and an increase in [3- and other structures. Our results also show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of calprotectin in the gut.     

Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point

Stem bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T_ms), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.