6M Guanidine Hydrochloride Research Articles

Expression, Purification, and Polyethylene Glycol Site-Specific Modification of Recombinant Human Interleukin 24 in Escherichia coli.

Interleukin 24 (IL-24) has a broad spectrum of specific antitumor activities without affecting normal cells. The recombinant human IL-24 (rhIL-24) expressed in E. coli has low biological activity due to lack of necessary glycosylation modification. In this study, based on the modification of the non-glycosylated IL-24 with polyethylene glycol (PEG), we aimed to improve the stability and prolong its half-life in vivo. Firstly, the recombinant plasmid containing the hIL-24 cDNA was prepared by the prokaryotic-expression plasmid pET-28a and transformed into E. coli BL21. After induced by isopropyl β-D-thiogalactoside (IPTG), the target protein rhIL-24 was expressed as insoluble inclusion body, which was solubilized and denatured by 6M guanidine hydrochloride. The denatured rhIL-24 was diluted to refold in the optimized buffer overnight at the protein concentration of 0.1mg/mL. The refolded rhIL-24 was mainly in the form of soluble aggregate, but high-purity monomer rhIL-24 was obtained through size exchange chromatography with the addition of SDS in elution buffer. The tertiary structure of rhIL-24 was confirmed by fluorescence spectroscopy. Western blot analysis showed that rhIL-24 could be site-specifically modified by mPEG5000-ALD. Methyl thiazolyl tetrazolium (MTT) assay showed no significant difference between mPEG5000-ALD-rhIL-24 and rhIL-24 in inhibiting the growth of melanoma cell line A375 in vitro. Pharmacokinetic studies showed that PEG modification could significantly improve the stability and prolong the half-life of rhIL-24 from 8.41 to 13.2h. The data strongly suggested that mPEG-ALD 5000 could site-specifically modify rhIL-24 expressed in E. coli. The PEG modification significantly prolonged the half-life of rhIL-24 without reducing its antitumor activity in vitro.

Lectin-binding and dissociation/reconstitution studies on the trichocysts of the dinoflagellate Oxyrrhis marina.

Trichocyst-enriched fractions were isolated from the dinoflagellate Oxyrrhis marina and subjected to protein staining and lectin-binding studies, to dissociation experiments using heat, and to solubilization/reconstitution experiments using 6M guanidine hydrochloride. The trichocysts could be stained by Alcian Blue and became labeled by the lectin Concanavalin A, but not by wheat germ agglutinin. The trichocysts did not dissociate when the fractions were heated for 5min at 40 or 50°C. Heating at 60°C resulted in the dissociation of trichocysts into irregular filamentous structures. These filaments were still present when the fractions were incubated for 5min at temperatures of 70 and 80°C. Reassembly was not achieved by subsequent cooling steps. The disintegration of trichocysts was also achieved in 6M guanidine hydrochloride, and reassembly into filamentous structures, similar to those obtained by heat, occurred after dialysis against distilled water. Electron microscopy revealed that the filaments created either by heat or using guanidine hydrochloride by far did not resemble native trichocysts. They were much thinner (5-7nm in width), missed the characteristic striation of electron-dense and -transparent lines along the longitudinal axes, and showed much more bending. Furthermore, they tend to merge to thicker shapeless structures and blob-like aggregates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that fractions enriched either in trichocysts or in reconstituted filamentous structures obtained in the guanidine hydrochloride solubilization experiments were dominated by proteins with relative molecular weights in the range of approximately 15 to 29kDa. Minor amounts of larger proteins were also detected. Tryptic in gel digestion followed by mass spectrometry confirmed the presence of almost the same set of proteins within the both, the trichocyst-enriched fractions, and the fractions of filaments reconstituted thereafter. These proteins were previously proposed to represent the matrix polypeptides of the trichocysts of Oxyrrhis marina (Rhiel et al., Protoplasma 255: 217-230, 2018).

BASELINE RECOVERY AND LONGITUDINAL STABILITY OF CEREBROSPINAL FLUID AMYLOID-β PEPTIDES: AN EVALUATION OF COLLECTION METHODS, LONGITUDINAL ASSAY PERFORMANCE, AND BIOMARKER STABILITY OVER A 2-YEAR PERIOD

Alzheimer's disease biomarker CSF Aβ1-42 is susceptible to numerous factors that compromise measurement accuracy. We evaluated assay stability, Aβ peptide recoveries, and Aβ peptide stabilities over a 2-year period to inform CSF handling and analysis protocols for clinical trials. A UP-RPLC-MS/MS assay was developed at Pharmaceutical Product Development with the Alzheimer's Association's Global Consortium for Biomarker Standardization to quantitate CSF Aβ1-42, 1-40, 1-38, 1-37, 1-34 using stable isotope-labeled peptides. CSF was collected from young normal controls and aged patients with dementia. CSF was denatured with 6M guanidine hydrochloride (GuHCl) at different stages of the collection and aliquoting procedure (after collection (“Collection”), before sub-aliquoting (“Aliquot”), or before solid phase extraction (“Analysis”)) and then stored at -80oC. In addition, a 1 mL aliquot was prepared in LoBind tubes at the time of collection, stored at -80oC, and GuHCl was added before extraction (“LoBind”). Measurements were made at baseline, and then every 3 months for 2 years. Longitudinal stabilities were assessed according to CLSI standards, with an acceptability cutoff of +/-10% of baseline. Earlier denaturation increased baseline CSF Aβ recovery relative to later denaturation (Mean (SD) CSF Aβ1-42collection=1061(476) pg/mL, CSF Aβ1-42Aliquot=996(414) pg/mL; vs. CSF Aβ1-42Analysis=822(385) pg/mL, CSF Aβ1-42LoBind=899(376) pg/mL (p<0.0002)). The Aβ1-42/Aβ1-40 ratio reduced recovery variability due to collection condition, and improved discrimination between control and dementia groups. The assay was stable for 2 years, but CSF Aβ peptide measurements and the Aβ1-42/Aβ1-40 ratio were not longitudinally stable for the study period. CSF Aβ1-42 was stable for 15months (“Collection”,“Aliquot”) or 18months (“Analysis”). The Aβ1-42/Aβ1-40 ratio was stable for 8 months (“Collection”,“Aliquot”) and 11months (“Analysis”). When measured with a UP-RPLC-MS/MS assay, earlier denaturation improved CSF Aβ peptide recovery. CSF Aβ1-42, Aβ1-40, Aβ1-38, Aβ1-34, and the Aβ1-42/Aβ1-40 ratio were not stable for the study period when samples were stored frozen. The Aβ1-42/Aβ1-40 ratio was useful for normalizing adsorption-related artifacts at baseline and for separating Dx groups, but was more sensitive to longitudinal instability. We recommend that UP-RPLC-MS/MS analysis of CSF Aβ peptides from frozen samples occur within the cutoffs specified or close to the time of collection.

Evaluation Activity of S-Layer Proteins and Filtrate of Lactobacillus Spp. against Some Pathogenic Microorganisms In Vitro

This project was conducted to evaluate the activity of S-layer proteins isolated from Lactobacillus in comparison with the activity of concentrated filtrate of Lactobacillus against some pathogenic microorganisms and against tumor cell lines in vitro. Twelve isolates of Lactobacillus spp. obtained from, vinegar, human milk, cow milk, yoghurt and vagina, wereused to detect the S-layer protein (Slp) by Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis(SDS-PAGE) then extracted it by excised the Slp pand and treated with 6M guanidine hydrochloride (G-HCl) to elute the protein from the gel. The Molecular weights (MW) of Slps were estimated between (37-63 kDa) depending on the Lactobacillus species. The concentrations of Slp were estimated by using a Kit based on the Biuret method. One isolate of each of Lactobacillus acidophilus and Lactobacillus casei, were selected depending on the MW and concentrations of S-layer proteins. The inhibitory effect of Lb. acidophilus and Lb. casei was determined against pathogenic microorganism; Pseudomonas aeruginosa , Escherichia coli, Staphylococcus aureus, Salmonella typhimurium,and Candida albicans on solid and liquid MRS media. Results revealed that less inhibitory activity against them was detected on the solid medium, compared to the liquid one. Greatest inhibitory effect of Lb. acidophilus and Lb. casei has appeared against P. aeruginosa when the zone of inhibition reached to 24 and 22 mm, respectively. It appeared that the inhibitory effect of Lb. acidophilus was more than that of Lb. casei against most of the tested microorganisms, while S-layer proteins have no effect against pathogenic microorganisms

Structural analyses combined with small-angle X-ray scattering reveals that the retention of heme is critical for maintaining the structure of horseradish peroxidase under denaturing conditions.

We analyzed the structure of horseradish peroxidase (HRP) under denaturing conditions of 9M urea or 6M guanidine hydrochloride (GdnHCl). Far-UV circular dichroism (CD) spectra indicated the existence of native-like secondary structure of holo-HRP in 9M urea. In addition, slight changes in near-UV and Soret region CD spectra of holo-HRP in 9M urea suggest that the tertiary structure of holo-HRP and the binding of heme remain partially intact in this condition. A transition in the thermal unfolding transition curve of holo-HRP in 9M urea indicated the existence of a considerable amount of secondary structure. However, no secondary structure, tertiary structure, or interaction between heme and HRP were observed in holo-HRP in 6M GdnHCl. Small-angle X-ray scattering indicated that although distal and proximal domains of holo-HRP in 9M urea might be partially unfolded, the central region that contains the heme might maintain its tertiary structure. Our results suggest that retention of the heme is essential for maintenance of the structure of HRP under highly denaturing conditions.

Studying the denaturation of bovine serum albumin by a novel approach of difference-UV analysis

A novel approach in the analysis of difference-UV spectrophotometric data for determining the heat denaturation degree of bovine serum albumin (BSA) was assessed. Five different parameters of difference-UV spectra were obtained by subtracting spectra of unheated and denatured protein solutions at different temperature-time combinations. BSA was found to exhibit a maximum degree of heat denaturation of about 17% compared to the complete unfolding caused by 6M guanidine hydrochloride. This low degree of heat denaturation is probably caused by the aggregation of the initially unfolded protein molecules. The kinetic analysis exhibited discontinuities in the Arrhenius plots, distinguishing the unfolding and aggregation phases of the denaturation process, whereas such a discrimination could not be obtained by differential scanning calorimetry analyses. The proposed method is accurate, fast, simple and sensitive enough to detect changes in the protein heat denaturation even at short temperature-time intervals.

Pcal_1699, an extremely thermostable malate dehydrogenase from hyperthermophilic archaeon Pyrobaculum calidifontis.

Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90°C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8M; however, in the presence of 4M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90% residual activity even after heating for 6h in boiling water.

Improved protocol to purify untagged amelogenin – Application to murine amelogenin containing the equivalent P70 → T point mutation observed in human amelogenesis imperfecta

Amelogenin is the predominant extracellular protein responsible for converting carbonated hydroxyapatite into dental enamel, the hardest and most heavily mineralized tissue in vertebrates. Despite much effort, the precise mechanism by which amelogenin regulates enamel formation is not fully understood. To assist efforts aimed at understanding the biochemical mechanism of enamel formation, more facile protocols to purify recombinantly expressed amelogenin, ideally without any tag to assist affinity purification, are advantageous. Here we describe an improved method to purify milligram quantities of amelogenin that exploits its high solubility in 2% glacial acetic acid under conditions of low ionic strength. The method involves heating the frozen cell pellet for two 15min periods at ∼70°C with 2min of sonication in between, dialysis twice in 2% acetic acid (1:250 v/v), and reverse phase chromatography. A further improvement in yield is obtained by resuspending the frozen cell pellet in 6M guanidine hydrochloride in the first step. The acetic acid heating method is illustrated with a murine amelogenin containing the corresponding P70→T point mutation observed in an human amelogenin associated with amelogenesis imperfecta (P71T), while the guanidine hydrochloride heating method is illustrated with wild type murine amelogenin (M180). The self-assembly properties of P71T were probed by NMR chemical shift perturbation studies as a function of protein (0.1–1.8mM) and NaCl (0–367mM) concentration. Relative to similar studies with wild type murine amelogenin, P71T self-associates at lower protein or salt concentrations with the interactions initiated near the N-terminus.

Chemical- and thermal-induced unfolding of Leishmania donovani ribose-5-phosphate isomerase B: a single-tryptophan protein.

Ribose-5-phosphate isomerase B (RpiB), a crucial enzyme of pentose phosphate pathway, was proposed to be a potential drug target for visceral leishmaniasis. In this study, we have analyzed the biophysical properties of Leishmania donovani RpiB (LdRpiB) enzyme to gain insight into its unfolding pathway under various chemical and thermal denaturation conditions by using fluorescence and CD spectroscopy. LdRpiB inactivation precedes the structural transition at lower concentrations of both urea and guanidine hydrochloride (GdHCl). 8-Anilinonapthalene 1-sulfonic (ANS) binding experiments revealed the presence of molten globule intermediate at 1.5M GdHCl and a nonnative intermediate state at 6-M urea concentration. Acrylamide quenching experiments further validated the above findings, as solvent accessibility of tryptophan residues increased with increase in GdHCl and urea concentration. The recombinant LdRpiB was completely unfolded at 6M GdHCl, whereas the enzyme molecule was resistant to complete unfolding even at 8-M urea concentration. The GdHCl- and urea-mediated unfolding involves a three-state transition process. Thermal-induced denaturation revealed complete loss of enzyme activity at 65°C with only 20% secondary structure loss. The formation of the well-ordered β-sheet structures of amyloid fibrils was observed after 55°C which increased linearly till 85°C as detected by thioflavin T dye. This study depicts the stability of the enzyme in the presence of chemical and thermal denaturants and stability-activity relationship of the enzyme. The presence of the intermediate states may have major implications in the way the enzyme binds to its natural ligand under various conditions. Also, the present study provides insights into the properties of intermediate entities of this important enzyme.

Further investigations on the polypeptides and reconstitution of prasinophycean ejectisomes

Ejectisome fragments were isolated from the prasinophyte Pyramimonas grossii and subjected to different treatments, i.e. Percoll density gradient centrifugation, incubation at pH 2.5 or at pH 10.8, or incubation in 6M guanidine hydrochloride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that Percoll density gradient centrifugation did not improve the purity of the ejectisome fragment-enriched fractions. The ejectisome fragments withstood pH 2.5 and pH 10.8 treatment, and no loosely bound polypeptides became detached. The disintegration of ejectisome fragments was achieved in 6M guanidine hydrochloride, and reassembly into filamentous, ejectisome-like structures occurred after dialysis against distilled water. Fractions enriched either in ejectisome fragments or in reconstituted ejectisome-like structures were dominated by three polypeptides with relative molecular weights of approximately 12.5–19kDa and two additional polypeptides of 23 and 26kDa. A polyclonal antiserum directed against an ejectisome fragment-enriched fraction weakly cross-reacted with these polypeptides, and no significant immuno-labelling of ejectisome fragments was registered. A positive immuno-label was achieved using immunoglobulin (IgG) fractions which were gained by selectively incubating nitrocellulose stripes of these polypeptides with the antiserum.

Biochemical and atomic force microscopic characterization of salmon nasal cartilage proteoglycan

Biological activities of salmon nasal cartilage proteoglycan fractions are known, however, structural information is lacking. Recently, the major proteoglycan of this cartilage was identified as aggrecan. In this study, global molecular images and glycosaminoglycan structure of salmon nasal cartilage aggrecan purified from 4M guanidine hydrochloride extract were analyzed using HPLCs and atomic force microscopy with bovine tracheal cartilage aggrecan as a control. The estimated numbers of sulfates per disaccharide unit of chondroitin sulfate chains of salmon and bovine aggrecans were similar (approximately 0.85). However, the disaccharide composition showed a higher proportion of chondroitin 6-sulfate units in salmon aggrecan, 60%, compared to 40% in bovine. Gel filtration HPLC and monosaccharide analysis showed the salmon aggrecan had a lower number (approximately one-third), but 1.5–3.3 times longer chondroitin sulfate chains than the bovine aggrecan. Atomic force microscopic molecular images of aggrecan supported the images predicted by biochemical analyses.

Modified CNBr Cleavage Protocol for Efficient Separation of Met-Ser Containing OmpX-Om14 Membrane Protein Fusion

Cleavage efficiencies of cyanogen bromide (CNBr) are drastically reduced in sequences wherein the methionine residue is succeeded by serine or threonine. Solubility issues associated especially with membrane proteins contribute further to reduced cleavage at Met-Xxx sites. Moreover, prolonged incubations of several hours to days, in highly acidic reaction conditions required for CNBr cleavage, often lead to protein degradation. To circumvent these problems, we have developed a protocol for CNBr cleavage of protein fusions, which is particularly useful for Met-Ser sequences. This protocol facilitates enhanced cleavage efficiencies in short incubation times of 1-2 hours. It incorporates up to 40% acetonitrile in 8M urea, or trace amounts of acetonitrile in 6M guanidine hydrochloride, in the final reaction. We demonstrate the successful application of this protocol for the Met-Ser containing membrane protein fusion OmpX-Om14. Cleavage of the Om14 (the yeast mitochondrial outer membrane protein) from E. coli OmpX (Outer membrane protein X) has provided yields of up to 70% product without detectable degradation levels. The direct use of the cleavage reaction for the rapid and cost-effective purification of Om14 has been shown. Using a database analysis, we discuss the importance of this protocol for several membrane proteins that have Met-Ser sequences

Designing of nitrile hydratase from alkaline protease using quanidine hydrochloride and cobalt metal ion

Alkaline protease (22.5kDa) from Bacillus licheniformis was denatured using 3–6M Guanidine hydrochloride followed by renaturation with cobalt metal ion in buffer solution (pH 7.5) in order to design cobalt dependent nitrile hydratase AP-xMGdn-Co, where x=3, 4, 5 and 6. The native alkaline protease showed a 20% residual molar ellipticity at 212nm while on treatment with Gdn-HCl, it got reduced to 12% due to nearly complete collapse of the globular structure of the protein. The observed circular dichroism spectra of the renatured proteases with Co metal ion are different from the native enzyme, because those molecules contain many metal chelating sites through its coordinating amino acid residues. The introduction of cobalt centered new active site was evidenced by the fluorescence spectroscopy (appearance of a new peak at 360nm) and ICP analysis (0.0476–0.0553mg/L). The specific activities of the modified enzymes were measured using the selective catalytic hydrolysis of 3-cyanopyridine to nicotinamide. The highest specific activity of renatured enzymes (AP-xMGdn-Co) were obtained at optimum pH of 7.5 and temperature of 50°C, due to the proper co-ordination of amino acid residue with cobalt metal ion and the appropriate arrangement of α-helix, β-strand, turn and unordered segment in the globular structure.

Stability of early-stage amyloid-β(1–42) aggregation species

Accumulation of aggregated amyloid-β protein (Aβ) is an important feature of Alzheimer's disease. There is significant interest in understanding the initial steps of Aβ aggregation due to the recent focus on soluble Aβ oligomers. In vitro studies of Aβ aggregation have been aided by the use of conformation-specific antibodies which recognize shape rather than sequence. One of these, OC antiserum, recognizes certain elements of fibrillar Aβ across a broad range of sizes. We have observed the presence of these fibrillar elements at very early stages of Aβ incubation. Using a dot blot assay, OC-reactivity was found in size exclusion chromatography (SEC)-purified Aβ(1–42) monomer fractions immediately after isolation (early-stage). The OC-reactivity was not initially observed in the same fractions for Aβ(1–40) or the aggregation-restricted Aβ(1–42) L34P but was detected within 1–2weeks of incubation. Stability studies demonstrated that early-stage OC-positive Aβ(1–42) aggregates were resistant to 4M urea or guanidine hydrochloride but sensitive to 1% sodium dodecyl sulfate (SDS). Interestingly, the sensitivity to SDS diminished over time upon incubation of the SEC-purified Aβ(1–42) solution at 4°C. Within 6–8days the OC-positive Aβ42 aggregates were resistant to SDS denaturation. The progression to, and development of, SDS resistance for Aβ(1–42) occurred prior to thioflavin T fluorescence. In contrast, Aβ(1–40) aggregates formed after 6days of incubation were sensitive to both urea and SDS. These findings reveal information on some of the earliest events in Aβ aggregation and suggest that it may be possible to target early-stage aggregates before they develop significant stability.

Ejectisins: tough and tiny polypeptides are a major component of cryptophycean ejectisomes

Fragments of discharged ejectisomes were isolated from two Cryptomonas and a Chroomonas species by detergent treatment followed by Percoll density gradient centrifugation. The fragments withstand high concentrated detergent solutions, reducing agents and freeze-thawing. Disintegration was achieved in 6M guanidine hydrochloride. Reassembly into long, filamentous, ejectisome-like structures occurred after dialysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the polypeptide patterns of isolated ejectisome fragments and of reconstituted ejectisome-like structures were dominated by polypeptides with relative molecular weights of approximately 6kDa. The polypeptides were not glycosylated and did not cross-react with antisera directed against recombinant Reb polypeptides which constitute the R-bodies of Caedibacter taeniospiralis. A polyclonal antiserum directed against reconstituted, ejectisome-like filaments cross-reacted with the 6-kDa polypeptides and immunolabeled extruded ejectisome filaments. Twenty amino acid residues, obtained by N-terminal amino acid sequence analysis, matched to polypeptide sequences deduced from cDNA sequences of the cryptophyte Guillardia theta. The term "ejectisins" is introduced for the 6-kDa polypeptides which represent a major component of cryptophycean ejectisomes.

Molecular weight distribution analysis by ultracentrifugation: Adaptation of a new approach for mucins

Mucins are the key macromolecular component of mucus, nature's natural lubricant, and one of the most important physical properties is their molecular weight distribution. A new approach for polydisperse polymers was recently published based on sedimentation velocity in the analytical ultracentrifuge and converts a distribution of sedimentation coefficient g(s) vs. s plot into a distribution of molecular weight utilising the power-law or scaling relationship between the sedimentation coefficient and molecular weight, s=κs Mwb where s is the sedimentation coefficient, Mw is the weight average molecular weight and κs and b are characteristic coefficients related to conformation. We investigate the possibility of using a large database of previously published values of s an M to define κs and b for both aqueous solution and aqueous solution supplemented by 6M guanidine hydrochloride (a solvent which helps to minimise sample degradation). These values are then applied to a study of the molecular weight distributions of preparations of human gastric mucin in the different solvents and at different stages of purification.

Recovery of active anti TNF-α ScFv through matrix-assisted refolding of bacterial inclusion bodies using CIM monolithic support

Anti TNF-α molecules are important as therapeutic agents for many of the autoimmune diseases in chronic stage. Here we report the expression and purification of a recombinant single chain variable fragment (ScFv) specific to TNF-α from inclusion bodies. In contrast to the conventional on column refolding using the soft gel supports, an efficient methodology using monolithic matrix has been employed. Nickel (II) coupled to convective interaction media (CIM) support was utilized for this purpose with 6M guanidine hydrochloride (GuHCl) as the chaotropic agent. The protein purified after solubilization and refolding proved to be biologically active with an IC50 value of 15μg. To the best of our knowledge, this is the first report showing the application of methacrylate based chromatographic supports for matrix-assisted refolding and purification of Escherichia coli inclusion bodies. The results are promising to elaborate the methodology further to exploit the potential positive features of monoliths in protein refolding science.

Comparison of activity and conformational changes of ficin during denaturation by urea and guanidine hydrochloride

The activity and conformational changes of ficin (EC 3.4.22.3), a cysteine protease from Ficus carica have been investigated during the denaturation by urea and guanidine hydrochloride (GuHCl). The denaturation of ficin was followed by activity measurements, fluorescence and circular dichroism (CD) spectroscopic studies. The enzyme activity decreased significantly at low concentration of both urea and GuHCl before unfolding of the enzyme molecule. The enzyme molecule was resistant for unfolding by urea under neutral conditions even at higher concentrations. However, the protein is susceptible to unfolding by urea at lower pH and transition follows a cooperative two-state rule with increasing concentration of urea. On the other hand, ficin molecule loses its complete structure in presence of 4M GuHCl under neutral conditions. The GuHCl-induced unfolding occurs in a simple two-state cooperative process. These results indicate the differential structural stability and fragility of active site of the enzyme towards denaturation by urea and GuHCl.

Effects of stretching on morphological and biochemical aspects of the extracellular matrix of the rat calcaneal tendon

Several studies have demonstrated the relationship between exercise and the extracellular matrix of muscle tendons, and have described alterations in their structural and biochemical properties when subjected to strenuous exercise. However, little is known about what happens to tendons when they are subjected to stretching. We evaluated the changes in the composition and structure of rat calcaneal tendons subjected to a stretching program. The animals had their muscles stretched for 30s with 30s of rest, with 10 repetitions, three and five times a week for 21days. For morphological analysis, the sections were stained with hematoxylin-eosin and toluidine blue. For biochemical analysis, the tendons were treated with 4M guanidine hydrochloride and analyzed in SDS-PAGE. The contents of total proteins and glycosaminoglycans were also measured. In the sections stained with toluidine blue, we could observe an increase of rounded cells, especially in the enthesis region. In the region next to the enthesis was a metachromatic region, which was more intensely stained in the stretched groups. In the tension regions, the cells appeared more aligned. Cellularity increased in both regions. The SDS-PAGE analysis showed a larger amount of collagen in the stretched groups and a polydispersed component of 65kDa in all the groups. The amounts of proteins and glycosaminoglycans were also larger in the stretched tendons. The agarose-gel electrophoresis confirmed the presence of dermatan sulfate in the tension and compression regions, and of chondroitin sulfate only in the latter. Our results showed that the stretching stimulus changed the cellularity and the amount of the extracellular matrix compounds, confirming that tendons are dynamic structures with a capacity to detect alterations in their load.

Conformational characterization of human eukaryotic initiation factor 2α: A single tryptophan protein

The α-subunit of the human eukaryotic initiation factor 2 (heIF2α), a GTP binding protein, plays a major role in the initiation of protein synthesis. During various cytoplasmic stresses, eIF2α gets phosphorylated by eIF2α-specific kinases resulting in inhibition of protein synthesis. The cloned and over expressed heIF2α, a protein with a single tryptophan (trp) residue was examined for its conformational characteristics using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The steady-state fluorescence spectrum, fluorescence lifetimes (τ1=1.13ns and τ2=4.74ns) and solute quenching studies revealed the presence of trp conformers in hydrophobic and differential polar environment at any given time. Estimation of the α-helix and β-sheet content showed: (i) more compact structure at pH 2.0, (ii) distorted α-helix and rearranged β-sheet in presence of 4M guanidine hydrochloride and (iii) retention of more than 50% ordered structure at 95°C. Hydrophobic dye binding to the protein with loosened tertiary structure was observed at pH 2.0 indicating the existence of a molten globule-like structure. These observations indicate the inherent structural stability of the protein under various denaturing conditions.