Gdn HCl Research Articles

Photolytic Cross-Linking to Probe Protein-Protein and Protein-Matrix Interactions in Lyophilized Powders.

Protein structure and local environment in lyophilized formulations were probed using high-resolution solid-state photolytic cross-linking with mass spectrometric analysis (ssPC-MS). In order to characterize structure and microenvironment, protein-protein, protein-excipient, and protein-water interactions in lyophilized powders were identified. Myoglobin (Mb) was derivatized in solution with the heterobifunctional probe succinimidyl 4,4'-azipentanoate (SDA) and the structural integrity of the labeled protein (Mb-SDA) confirmed using CD spectroscopy and liquid chromatography/mass spectrometry (LC-MS). Mb-SDA was then formulated with and without excipients (raffinose, guanidine hydrochloride (Gdn HCl)) and lyophilized. The freeze-dried powder was irradiated with ultraviolet light at 365 nm for 30 min to produce cross-linked adducts that were analyzed at the intact protein level and after trypsin digestion. SDA-labeling produced Mb carrying up to five labels, as detected by LC-MS. Following lyophilization and irradiation, cross-linked peptide-peptide, peptide-water, and peptide-raffinose adducts were detected. The exposure of Mb side chains to the matrix was quantified based on the number of different peptide-peptide, peptide-water, and peptide-excipient adducts detected. In the absence of excipients, peptide-peptide adducts involving the CD, DE, and EF loops and helix H were common. In the raffinose formulation, peptide-peptide adducts were more distributed throughout the molecule. The Gdn HCl formulation showed more protein-protein and protein-water adducts than the other formulations, consistent with protein unfolding and increased matrix interactions. The results demonstrate that ssPC-MS can be used to distinguish excipient effects and characterize the local protein environment in lyophilized formulations with high resolution.

Existence of Different Structural Intermediates and Aggregates on the Folding Pathway of Ovalbumin

Structural modifications of ovalbumin in presence of different concentration of guanidine hydrochloride (Gdn HCl) and glucose were investigated by using intrinsic fluorescence, Fourier transform infra-red spectroscopy, circular dichroism and 8-anilino-1-naphthalene-sulphonic acid, to confirm that partially folded intermediates of ovalbumin lead to aggregation. The two partially folded intermediates of ovalbumin were observed one at 1M Gdn HCl and another in the presence of 20mM glucose at 3M Gdn HCl. Both intermediates exist as compact states with altered intrinsic fluorescence, prominent β-sheet secondary structure and enhanced ANS binding. Ovalbumin in the presence of glucose required more concentration of Gdn HCl (3M) to exist as an intermediate state than control (1M). Such alpha-helix/beta-sheet transition of proteins is a crucial step in amyloidogenic diseases and represents an internal rearrangement of local contacts in an already folded protein. Further, incubation for 24h resulted in the formation of aggregates as detected by thioflavin T-assay. On further increasing the concentration of glucose to 50mM and incubation time for various days resulted in the formation of molten globule state of ovalbumin at 6th day. Later on, at 10th day advanced glycated end products were observed.

Purification and biochemical characterization of a serine proteinase inhibitor from Derris trifoliata Lour. seeds: Insight into structural and antimalarial features

A potent serine proteinase inhibitor was isolated and characterized from the seeds of the tropical legume liana, Derris trifoliata (DtTCI) by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. SDS-PAGE as well as MALDI-TOF analysis showed that DtTCI is a single polypeptide chain with a molecular mass of approximately 20 kDa. DtTCI has three isoinhibitors (pI: 4.55, 5.34 and 5.72) and, inhibited both trypsin and chymotrypsin in a 1:1 molar ratio. Both Dixon plots and Lineweaver-Burk double reciprocal plots revealed a competitive inhibition of trypsin and chymotrypsin activity, with inhibition constants (K(i)) of 1.7x10(-10) and 1.25x10(-10) M, respectively. N-terminal sequence of DtTCI showed over 50% similarity with numerous Kunitz-type inhibitors of the Papilionoideae subfamily. High pH amplitude and broad temperature optima were noted for DtTCI, and time course experiments indicated a gradual loss in inhibitory potency on treatment with dithiothreitol (DTT). Circular Dichroism (CD) spectrum of native DtTCI revealed an unordered structure whereas exposure to thermal-pH extremes, DTT and guanidine hydrochloride (Gdn HCl) suggested that an abundance of beta-sheets along with intramolecular disulfide bonds provide conformational stability to the active site of DtTCI, and that severity of denaturants cause structural modifications promoting inhibitory inactivity. Antimalarial studies of DtTCI indicate it to be a potent antiparasitic agent.

Fibril Formation of hsp10 Homologue Proteins and Determination of Fibril Core Regions: Differences in Fibril Core Regions Dependent on Subtle Differences in Amino Acid Sequence

Heat shock protein 10 (hsp10) is a member of the molecular chaperones and works with hsp60 in mediating various protein folding reactions. GroES is a representative protein of hsp10 from Escherichia coli. Recently, we found that GroES formed a typical amyloid fibril from a guanidine hydrochloride (Gdn–HCl) unfolded state at neutral pH. Here, we report that other hsp10 homologues, such as human hsp10 (Hhsp10), rat mitochondrial hsp10 (Rhsp10), Gp31 from T4 phage, and hsp10 from the hyperthermophilic bacteria Thermotoga maritima, also form amyloid fibrils from an unfolded state. Interestingly, whereas GroES formed fibrils from either the Gdn–HCl unfolded state (at neutral pH) or the acidic unfolded state (at pH 2.0–3.0), Hhsp10, Rhsp10, and Gp31 formed fibrils from only the acidic unfolded state. Core peptide regions of these protein fibrils were determined by proteolysis treatment followed by a combination of Edman degradation and mass spectroscopy analyses of the protease-resistant peptides. The core peptides of GroES fibrils were identical for fibrils formed from the Gdn–HCl unfolded state and those formed from the acidic unfolded state. However, a peptide with a different sequence was isolated from fibrils of Hhsp10 and Rhsp10. With the use of synthesized peptides of the determined core regions, it was also confirmed that the identified regions were capable of fibril formation. These findings suggested that GroES homologues formed typical amyloid fibrils under acidic unfolding conditions but that the fibril core structures were different, perhaps owing to differences in local amino acid sequences.

Evidence that endothelial lipase remodels high density lipoproteins without mediating the dissociation of apolipoprotein A-I

Endothelial lipase (EL) is a triglyceride lipase gene family member that has high phospholipase and low triglyceride lipase activity. The aim of this study was to determine whether the phospholipase activity of EL is sufficient to remodel HDLs into small particles and mediate the dissociation of apolipoprotein A-I (apoA-I). Spherical, reconstituted HDLs (rHDLs) containing apoA-I only [(A-I)rHDLs], apoA-II only [(A-II)rHDLs], or both apoA-I and apoA-II [(A-I/A-II) rHDLs] were prepared. The rHDLs, which contained only cholesteryl esters in their core and POPC on the surface, were incubated with EL. As the rHDLs did not contain triacylglycerol, only the POPC was hydrolyzed. Hydrolysis was greater in the (A-I/A-II)rHDLs than in the (A-I)rHDLs. The (A-II)rHDL phospholipids were not hydrolyzed by EL. EL remodeled the (A-I)rHDLs and (A-I/A-II)rHDLs, but not the (A-II)rHDLs, into smaller particles. The reduction in particle size was related to the amount of phospholipid hydrolysis, with the diameter of the (A-I/A-II)rHDLs decreasing more than that of the (A-I)rHDLs. These changes did not affect the conformation of apoA-I, and neither apoA-I nor apoA-II dissociated from the rHDLs. Comparable results were obtained when human plasma HDLs were incubated with EL. These results establish that the phospholipase activity of EL remodels plasma HDLs and rHDLs into smaller particles without mediating the dissociation of apolipoproteins.

Low molecular mass pectate lyase from Fusarium moniliforme: similar modes of chemical and thermal denaturation

A low molecular mass pectate lyase from Fusarium moniliforme was unfolded reversibly by urea and Gdn–HCl at its optimum pH of 8.5, as monitored by intrinsic fluorescence, circular dichroism, and enzymatic activity measurements. Equilibrium unfolding studies yielded a Δ G(H 2O) of 1.741 kcal/mol, D 1/2 of 2.3 M, and m value of 0.755 kcal/mol M with urea and a Δ G(H 2O) of 1.927 kcal/mol, D 1/2 of 1.52 M, and m value of 1.27 kcal/mol M with Gdn–HCl as the denaturant. Thermal denaturation of the pectate lyase at, pH 8.5, was also reversible even after exposure to 75 °Cfor 10 min. Thermodynamic parameters calculated from thermal denaturation curves at pH values from 5.0 to 8.5 yielded a Δ C p of 0.864 kcal/(mol K). The Δ G(25 °C) at, pH 8.5, was 2.06 kcal/mol and was in good agreement with the Δ G(H 2O) values obtained from chemical denaturation curves. There was no exposure of hydrophobic pockets during chemical or thermal denaturation as indicated by the inability of ANS to bind the pectate lyase.

Amino Acids Conserved at the C-Terminal Half of the Ribonuclease T2 Family Contribute to Protein Stability of the Enzymes

The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr(101), Phe(102), Ala(105), and Phe(190) resulted in a significant decrease in themostability; the T(m) values were 47-58 degrees C compared to that for the wild type (64 degrees C). Mutations of Pro(125), Gly(127), Gly(144), and Val(165) caused a moderate decrease in thermostability (T(m): 60-62 degrees C). In contrast, mutations of Asp(107) and Gly(173) did little effect on thermostability. The contribution of Tyr(101), Phe(102), Pro(125), and Gly(127) to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr(101), Phe(102), Ala(105), Pro(125), Gly(127), Gly(144), Leu(162), Val(165), and Phe(190) conserved in the RNase T2 family play an important role in the stability of the proteins.

Acid-Induced Partly Folded Conformation Resembling a Molten Globule State of Xylanase from an Alkalothermophilic Bacillus sp

Nonnative protein structures having a compact secondary, but not rigid tertiary structure, have been increasingly observed as intermediate states in protein folding. We have shown for the first time during acid-induced unfolding of xylanase (Xyl II) the presence of a partially structured intermediate form resembling a molten globule state. The conformation and stability of Xyl II at acidic pH was investigated by equilibrium unfolding methods. Using intrinsic fluorescence and CD spectroscopic studies, we have established that Xyl II at pH 1.8 (A-state) retains the helical secondary structure of the native protein at pH 7.0, while the tertiary interactions are much weaker. At variance, from the native species (N-state), Xyl II in the A-state binds 1-anilino-8-sulfonic acid (ANS) indicating a considerable exposure of aromatic side chains. Lower concentration of Gdn HCl are required to unfold the A-state. For denaturation by Gdn HCl, the midpoint of the cooperative unfolding transition measured by fluorescence for the N-state is 3.5 ± 0.1 M, which is higher than the value (2.2 ± 0.1 M) observed for the A-state at pH 1.8. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its structural stability that is characteristic for native proteins.

Conformational Features of a Truncated Staphylococcal Nuclease R (SNR135) and Their Implications for Catalysis

Conformational features of a truncated (14 amino acid residues deleted from the C-terminus) staphylococcal nuclease R (SNR135) and the ternary complex of SNR135–Ca2+–pdTp were studied using circular dichroism (CD) spectra and 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence spectra under different conditions. Kinetic parameters such asKDNAM,KDNAS,KCaM,KCaA, andKpdTpdof SNR135 were also determined. The results show that SNR135 contains some residual secondary structure and some tertiary structure elements as indicated by far-UV and near-UV CD spectra and that it has the ability to fold into a native-like state in the presence of pdTp and Ca2+, but there are obvious differences both in secondary structure and in tertiary structure between the SNR135–Ca2+–pdTp complex and SNase R. The unfolding curves in Gdn–HCl show that the stability of the native-like conformation of the SNR135–Ca2+–pdTp complex is much less than that of SNase R though the ligand (Ca2+, pdTp) binding increases the stability of the SNR135–Ca2+–pdTp complex to some extent. Comparison of the kinetic parameters of SNR135 with those of the full-length nuclease shows that both SNR135 and SNase R have the same value ofKpdTpdand very similar values ofKCaMandKCaA, but SNR135 has larger values ofKDNAMandKDNASthan SNase R. Such results indicate that the C-terminal deletion for SNR135 does not greatly affect the ligand (Ca2+, pdTp) binding and decreases the binding affinity of the DNA substrate to the nuclease, implying that the amino acid residues at the ligand binding sites in SNR135 are probably arranged in a similar topology to those in SNase R and that effective binding of the DNA substrate to the enzyme needs the conformational integrity of the entire enzyme molecule. Furthermore, it is suggested that the binding sites of pdTp and DNA substrate may overlap but are not exactly the same. This paper also provides evidence obtained by monitoring ANS-binding fluorescence that the partially unfolded conformation of SNR135 is not in the molten globule state.

Degradation of recombinant porcine growth hormone in the presence of guanidine hydrochloride

This study was conducted to investigate the effect of guanidine hydrochloride (Gdn HCl) on the rate of chemical degradation of recombinant porcine growth hormone (pGH) at alkaline pH. The protein was incubated in 0.2 M Tris buffer, pH 9 at 37°C containing Gdn HCl at concentrations ranging from 0 to 6 M. The conformation of pGH under the different solution conditions was assessed by circular dichroism spectropolarimetry. Degradation was monitored by reversed phase HPLC (RP-HPLC), size exclusion chromatography (SEC) and SDS-PAGE. An associated form of pGH was populated in the presence of 3 and 4 M Gdn HCl, whereas the protein was completely unfolded in 5 and 6 M Gdn HCl. The apparent degradation rate constant determined by RP-HPLC increased by a factor of 18 in the presence of 3 M Gdn HCl and by a factor of five in the presence of 6 M Gdn HCl relative to that in buffer alone. RP-HPLC and SEC data suggested that the product distribution also differed between buffer and solutions containing Gdn HCl. While the increased rate of degradation for the denatured state was not unexpected, the dramatic increase at an intermediate Gdn HCl concentration was surprising and indicated the importance of considering chemical instability when proteins are exposed to denaturants.

Structural and Functional Role of Tryptophan in Xylanase from an ExtremophilicBacillus:Assessment of the Active Site

Microenvironment and conformation of the active site of xylanase from an extremophilicBacilluswas deciphered for the first time using fluorescence spectroscopy. NBS modified enzyme showed complete inactivation and the kinetic analysis implicated the presence of an essential tryptophan at the active site of xylanase. Xylan (0.5%) protected the enzyme completely from inactivation with NBS, whereas it afforded 35% protection against the loss of fluorescence, suggesting that not all the tryptophans are involved at the substrate binding site. Quenching studies revealed that acrylamide was more efficient than KI and CsCl as indicated by the higher Stern–Volmer quenching constants (Ksv). The steric factor represented by the percentage accessibility of the tryptophan residues of XylII was higher with the positively charged Cs+(80) than with the negatively charged I−(10), suggesting that the tryptophan residues are located in a relatively electronegative environment. In the presence of 6 M Gdn HCl the fluorescence shifted to 350 nm with increased accessibility of the fluorophore to the quenchers. The proximity of the essential carboxyl groups with a high pKa value of 6.9 [Chauthaiwale and Rao (1994)Biochim. Biophys. Acta] probably contributes to the electronegative environment of the tryptophan residue. Our results on sequence analysis of the gene encoding for XylII (Accession Number U83602 in the GenBank database) have shown that Trp 61 is highly conserved and may play a role in the structure–function relationship of the enzyme.

Denaturation of horseradish peroxidase with urea and guanidine hydrochloride

Favourable effects of urea and guanidine hydrochloride (Gdn HCI) on solubilization of the polar, non-polar and peptide groups of horseradish peroxidase (HRP), an example of a globular protein, provide the driving force for unfolding of HRP, in a reversible two-state process. The intrinsic or conformational stability of HRP at various pH values and temperatures has been estimated by the linear extrapolation method (LEM), a denaturant binding model (DBM) and Tanford's model. There is good agreement between these methods. Tanford's model shows that urea interacts with non-polar groups to a greater extent than Gdn HCl does, whereas Gdn HCI interacts more effectively with the peptide groups of HRP.

Reduction of Disulfide Bonds in Proteins by 2-Aminothiophenol under Weakly Acidic Conditions

We developed a method for reducing disulfide bonds in proteins under weakly acidic conditions by use of 2-aminothiophenol. The disulfide bonds in hen egg-white lysozyme, ribonuclease A, and soybean trypsin inhibitor were quantitatively reduced by 2-aminothiophenol in phosphate buffer, pH6, containing 8 M Gdn HCl, 1 mM EDTA, and 20% ethanol, for 60 min at 40 degrees C. On analysis of the RP-HPLC patterns of tryptic peptides, which were derived from reduced and S-alkylated lysozyme and ribonuclease A at pH 6, it was confirmed that no side reaction occurred. Moreover, the reduction under weakly acidic conditions was demonstrated to be applicable for the location of such a labile residue as O-acetylated tyrosine.

Hydrophobic interaction chromatography of fibroblast proteoglycans.

We have investigated the hydrophobic properties of human skin fibroblast proteoglycans and related material by affinity chromatography on Octyl-Sepharose CL-4B in 4 M guanidinium hydrochloride (GdnHCl). Proteoglycans and related material could be separated into non-, medium and highly hydrophobic forms by elution with gradients of Triton X-100 in 4 M Gdn HCl. The non-hydrophobic material included endogenously produced glycosaminoglycan chains and oligosaccharides as well as an HS-proteoglycan with a 35 kDa core. The 65-70 kDa core (glypican-related) proteoglycans appeared among the highly hydrophobic ones, but variable proportions were seen both in the medium and the non-hydrophobic material. Other membrane-bound proteoglycans, like fibroglycan (45 kDa core) and the HS-proteoglycans with 90 and 130 kDa cores, as well as the CS/DS-proteoglycan with a 90 kDa core, were all of high hydrophobicity. There were also indications of a highly hydrophobic CS/DS-proteoglycan with a 45 kDa core. The extracellular proteoglycans, PG-L, PG-S1 and PG-S2, and the HS-proteoglycans with 350 and 250 kDa cores were all of medium hydrophobicity. These proteoglycans emerged in distinct positions when the column was eluted with a gradient of 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate.

The competitive inhibition of Trichoderma reesei C30 cellobiohydrolase I by guanidine hydrochloride

The P-nitrophenylcellobiosidase (PNPCase) activity of Trichoderma reesei cellobiohydrolase I (CBH I) was competitively inhibited by concentrations of guanidine hydrochloride (Gdn HC1) that did not affect the tryptophan fluorescence of this enzyme. The K m of CBH I, 3.6 mM, was increased to 45.4 mM in the presence of 0.14 M Gdn HCl, the concentration that was required to inhibit the enzyme by 50%. A similar concentration of lithium chloride and urea had little effect on the PNPCase activity of CBH I. Maximal inhibition was pH dependent, occurring in the range of pH 4.0 to 5.0, which is in the range for maximal activity. Analysis of the inhibition data indicated that 1.2 molecules of Gdn HCl combine reversibly with I molecule of CBH I. Other hydrolases and proteases were also inhibited by Gdn HCl. It is suggested that the inhibition of CBH I by Gdn HCl occurs as a result of the interaction between the positively charged guanidinium group of Gdn HCl and the carboxylate group of glutamic acid 126, postulated to be in the catalytic center of this enzyme.

The role of noncovalent forces in micelle formation by vicilin from Vicia faba. III. The effect of urea, guanidine hydrochloride and sucrose on protein interactions

The effect of denaturing (urea, guanidine hydrochloride (Gdn HCl)), and stabilizing (sucrose) media on the capacity of vicilin from fababeans to self-associate into a micelle arrangement was examined. The micelle observations were related to protein conformational parameters, specifically thermal stability plus aliphatic (CPA) and aromatic (ANS) surface hydrophobicity (S 0). Both urea and GdnHCl had a negative impact on micelle formation at low concentrations (0·1 m urea and 0·2 m GdnHCl). At higher denaturant levels, the deteriorating micelle response was correlated with a decrease in thermal stability (T d, ΔH) and a decrease in aromatic and aliphatic S 0 values. Exposure to sucrose was also detrimental to micelle formation; this was attributed to preferential hydration of the protein molecules. In all situations, a delicate balance of hydrophilic-hydrophobic forces seemed to be required for the micelle phenomenon.

Proteoglycans from squid cranial cartilage: extraction and characterization

Abstract 1. 1. The majority of proteoglycans (85% of uronic acid) were extracted from squid cranial cartilage with 2% SDS. 2. 2. Conventional methods with 4 M Gdn HCl alone or with 4% Zwittergent, extracted only 30%. 3. 3. The proteoglycans were isolated by density gradient centrifugation. 4. 4. At least 25% of the extracted proteoglycans with 2% SDS do not form aggregates with hyaluronate. 5. 5. The proteoglycan monomers are of varying hydrodynamic size, those extracted with 4 M Gdn HCl being the smallest of average molecular weight 750,000.

Subunit structure and dissociation of Callinectes sapidus hemocyanin

The hemocyanin of the blue crab, Callinectes sapidus has two major components sedimenting with approximate sedimentation coefficients of 17 S and 25 S. Molecular weight data based on light scattering and sedimentation equilibrium measurements at pH 7.8 suggest that the two components have molecular weights of approximately 450 000 and 900 000 in the presence of stabilizing Ca 2+. In the absence of Ca 2+, the molecular weights are found to be about 5% lower, suggesting some dissociation of the hemocyanin components. Circular dichroism and optical rotatory dispersion measurements in the far-ultraviolet region gave nearly identical spectra for the two components. Based on the reference parameters of Chen et al. (Chen, Y.H., Yang, J.T. and Martinez, H.M. (1972) Biochemistry 11, 4120—4131 and Chen, Y.H., Yang, J.T. and Chan, K.H. (1974) Biochemistry 13, 3350–3359), estimates of 16–20% α-helix, 40–60% β-structure, and 30–40% random organization were obtained for the two hemocyanin components. Exposure to 6 M Gdn HCl gave light scattering molecular weights of approx. 68 000 and 77 000, which is close to one-sixth of the molecular weight of the 17 S component. These results support the view that the two components of C. sapidus hemocyanin share the hexameric and dodecameric organization common to arthropod hemocyanins. The salts of the Hofmeister series and the ureas are found to dissociate the dodecameric component with the former exhibiting the usual order of effectiveness of NaCl, NaBr, NaI, and NaClO 4 dissociation, while the ureas show an inverse order of decreasing effectiveness in going from urea to methyl-, ethyl- and propylurea. This suggests that polar and ionic interactions are relatively more important than hydrophobic interactions for the stabilization of the dodecameric form of C. sapidus hemocyanin. The dissociation behavior of the 17 S hexameric species by GdnHCl in the 0–1.5 M concentration region (where essentially no denaturation occurs), based on light scattering molecular-weight measurements, is satisfactorily accounted for by equations describing the dissociation of hexamers to monomers.