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Background: Targeted drug delivery is a novel method to specifically deliver anticancer therapeutics to tumor sites. Gonadotropin-releasing hormone (GnRH) is a decapeptide, and its target binding property has attracted attention as a means of targeted drug delivery. Human pancreatic ribonuclease 1 (hpRNase1) has been shown to exert anticancer properties, when fused to a targeting moiety. The goal of the present study was to add a GnRH targeting peptide to the N-terminus of hpRNase1 to specifically target GnRH receptor (GnRH-R) expressing cells., Methods: This in vitro study was conducted at Shiraz Institute for Cancer Research (Shiraz, Iran) in 2019. The coding sequence of GnRH and hpRNase1 were fused, and the chimeric protein together with non-fused hpRNase1 were produced in E. coli (BL21). The recombinant proteins were purified, and their biological activity was evaluated using MTT and apoptosis assays. Non-parametric Kruskal-Wallis tests with Dunn's post hoc tests were performed to determine the significant differences between the study groups., Results: GnRH-hpRNase1 chimeric protein specifically inhibited the proliferation of PC-3 (P=0.021), LNCaP (P=0.034), and AD-Gn (P=0.041) cells, while the growth of negative cells (AD-293) was not significantly affected (P=0.081). GnRH-hpRNase1 decreased the IC50 values more than non-fused hpRNase1, by approximately 26.5-fold (P=0.036) for PC-3 cells, and exerted its growth inhibitory effects through apoptosis induction., Conclusion: Fusion of GnRH to hpRNase1 structure produced an enzyme, which could specifically target tumor cells. This approach can be used to eliminate tumors that harbor GnRH-R., (Copyright: © Iranian Journal of Medical Sciences.)

Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection., Competing Interests: The University of Maryland, Baltimore County (UMBC) has filed and has had issued several patents related to microwave-based lysing and DNA fragmentation. Those patents are licensed to Lyse-It LLC, a Maryland-based biotechnology company, which Professor Geddes currently holds stock. Patent #: US US9500590B2. “Assays for pathogen detection using microwaves for lysing and accelerating metal-enhanced fluorescence.” Patent #: US10294451B2 “Flow and static lysing systems and methods for ultrarapid isolation and fragmentation of biological materials by microwave irradiation”, Chris D. Geddes. Lyse-It LLC was not involved in the study design of the material within this paper, nor do any of the author receive any salaries or consultancies from Lyse-It LLC. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Purpose:: To investigate the effect of nicotinamide on the secretion of pro-an giogenic and pro-inflammatory cytokines in uveal melanoma cell lines., Methods:: Two human uveal melanoma cell lines (92.1 and OCM-1) were treated with nicotinamide (10 mmol/L) or control media for 48 hours in culture. The su perna tant from each culture was used in sandwich enzyme-linked immuno sorbent assay-based angiogenesis and inflammation arrays to evaluate the effects of exogenously administered nicotinamide on the secretion of a total of 20 pro-an gio genic and pro-inflammatory proteins., Results:: Seven pro-angiogenic cytokines were detected under control conditions for both uveal melanoma cell lines. Treatment with nicotinamide resulted in a significant decrease in secretion of the following pro-angiogenic cytokines: angiogenin, angiopoietin-2, epidermal growth factor, and vascular epithelial growth factor-A in the 92.1 cells; basic fibroblast growth factor in the OCM-1 cells; and placenta growth factor in both cell lines. Among the pro-inflammatory proteins, monocyte chemotactic protein-1 and interleukin-8 were expressed in both untreated cell lines and both were significantly reduced when treated with nicotinamide., Conclusions:: Results from this in vitro model suggest that nicotinamide may have anti-inflammatory and anti-angiogenic properties, which may open the possibility of using it as a chemopreventive agent for uveal melanoma; however, further studies including animal models are warranted.

To date, little work has been conducted on the relationship between solute and buffer molecules and conformational exchange motion in enzymes. This study uses solution NMR to examine the effects of phosphate, sulfate, and acetate in comparison to MES- and HEPES-buffered references on the chemical shift perturbation and millisecond, chemical, or conformational exchange motions in the enzyme ribonuclease A (RNase A), triosephosphate isomerase (TIM) and HisF. The results indicate that addition of these solutes has a small effect on (1)H and (15)N chemical shifts for RNase A and TIM but a significant effect for HisF. For RNase A and TIM, Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, however, show significant solute-dependent changes in conformational exchange motions. Some residues show loss of millisecond motions relative to the reference sample upon addition of solute, whereas others experience an enhancement. Comparison of exchange parameters obtained from fits of dispersion data indicates changes in either or both equilibrium populations and chemical shifts between conformations. Furthermore, the exchange kinetics are altered in many cases. The results demonstrate that common solute molecules can alter observed enzyme millisecond motions and play a more active role than what is routinely believed.

Background: Age-related macular degeneration (AMD) is currently the leading cause of blindness in developed countries. Bevacizumab is a widely used anti-VEGF agent that is a commonly applied therapy for neovascular AMD; however, a consequence of bevacizumab therapy may be the activation of compensatory angiogenic signalling. Combination of bevacizumab with 3,4 dihydroxyphenyl ethanol (DPE) may attenuate this compensatory signalling. The goal of the study was to investigate this therapeutic option in a human retinal pigment epithelial cell line (ARPE-19)., Methods: ARPE-19 cells were incubated under both normoxic and hypoxic conditions. The cells were treated as follows: control, 100 µM DPE, 0.25 mg/ml bevacizumab, the combination of DPE and bevacizumab. Media was harvested after 24 h for sandwich ELISA-based angiogenesis assays. The secretion of the following 10 pro-angiogenic cytokines was measured: angiogenin, ANG2, EGF, bFGF, HB-EGF, PDGF-BB, Leptin, PIGF, HGF, and VEGF-A., Results: Treatment of ARPE-19 cells with bevacizumab significantly increased the secretion of angiogenin. Secretion of angiogenin and VEGF-A were significantly reduced following treatment with DPE under both normoxia and hypoxia. In addition, angiogenin secretion was significantly reduced following treatment with the combination of DPE and bevacizumab compared to bevacizumab alone., Conclusions: Compensatory angiogenic signalling may occur in neovascular AMD following treatment with bevacizumab. Here we show that DPE, both alone and in combination with bevacizumab, can reduce the secretion of angiogenin, a cytokine that has been upregulated following treatment with bevacizumab in RPE cells. Therefore, DPE may represent a possible therapeutic agent to be used in combination with bevacizumab for the treatment of neovascular AMD.

Kidney cells of animals including human and marine invertebrates contain high amount of the protein denaturant, urea. Methylamine osmolytes are generally believed to offset the harmful effects of urea on proteins in vitro and in vivo. In this study we have investigated the possibility of glycine to counteract the effects of urea on three proteins by measuring thermodynamic stability, ΔGD° and functional activity parameters (K(m) and k(cat)). We discovered that glycine does not counteract the effects of urea in terms of both protein stability and functional activity. We also observed that the glycine alone is compatible with enzymes function and increases protein stability in terms of T(m) (midpoint of thermal denaturation) to a great extent. Our study indicates that a most probable reason for the absence of a stabilizing osmolyte, glycine in the urea-rich cells is due to the fact that this osmolyte is non-protective to macromolecules against the hostile effects of urea, and hence is not chosen by evolutionary selection pressure.

We have recently concluded from the heat-induced denaturation studies that polyols do not affect deltaG(D) degrees (the Gibbs free energy change (deltaG(D)) at 25 degrees C) of ribonuclease-A and lysozyme at physiological pH and temperature, and their stabilizing effect increases with decrease in pH. Since the estimation of deltaG(D) degrees of proteins from heat-induced denaturation curves requires a large extrapolation, the reliability of this procedure for the estimation of deltaG(D) degrees is always questionable, and so are conclusions drawn from such studies. This led us to measure deltaG(D) degrees of ribonuclease-A and lysozyme using a more accurate method, i.e., from their isothermal (25 degrees C) guanidinium chloride (GdmCl)-induced denaturations. We show that our earlier conclusions drawn from heat-induced denaturation studies are correct. Since the extent of unfolding of heat- and GdmCl-induced denatured states of these proteins is not identical, the extent of stabilization of the proteins by polyols against heat and GdmCl denaturations may also differ. We report that in spite of the differences in the structural nature of the heat- and GdmCl-denatured states of each protein, the extent of stabilization by a polyol is same. We also report that the functional dependence of deltaG(D) of proteins in the presence of polyols on denaturant concentration is linear through the full denaturant concentration range. Furthermore, polyols do not affect the secondary and tertiary structures of the native and GdmCl-denatured states.

Thermal denaturation curves of lysozyme and ribonuclease-A were determined by measuring their far-UV circular dichroism (CD) spectra in the presence of different concentrations of five polyols (sorbitol, glycerol, mannitol, xylitol and adonitol) at various pH values in the range 7.0--1.9. The denaturation curve at each polyol concentration and pH was analysed to obtain values of T(m) (midpoint of denaturation) and DeltaH(m) (enthalpy change at T(m)), and these DeltaH(m) and T(m) values obtained at different pH values were used to obtain DeltaC(p) (constant-pressure heat capacity change) at each polyol concentration. Using values of DeltaH(m), T(m) and DeltaC(p) in the Gibbs-Helmholtz equation, DeltaG(D) degrees (Gibbs energy change at 25 degrees C) was determined at a given pH and polyol concentration. Main conclusions of this study are that polyols have no significant effect on DeltaG(D) degrees at pH 7.0, and they stabilise proteins in terms of DeltaG(D) degrees against heat denaturation at lower pH values. Other conclusions of this study are: (i) T(m) at each pH increases with increasing polyol concentration, (ii) DeltaH(m) remains, within experimental error, unperturbed in the presence of polyols, and (iii) DeltaC(p) depends on polyol concentration. Furthermore, measurements of the far- and near-UV CD spectra suggested that secondary and tertiary structures of both proteins in their native and denatured states are not perturbed on the addition of polyols.

The thermal denaturation of ribonuclease A has been studied by differential scanning calorimetry in the presence of 4-chlorobutan-1-ol. The thermal transitions were observed to be reversible at pH 5.5 in the presence of low concentration (up to 50 mM) of the alcohol, irreversible in the intermediate (50 mM < c < mM) and again reversible in the presence of 250 mM and higher concentrations of 4-chlorobutan-1-ol. In the presence of 50 mM 4-chlorobutan-1-ol, ribonuclease A is present in two conformational states unfolding at different temperatures. The reversible thermal transitions have been fitted to a two-state native-to-denatured mechanism. Irreversible thermal transitions have been analyzed according to two-state irreversible native-to-denatured kinetic model. Using the irreversible model, rate constant as a function of temperature and energy of activation of the irreversible process have been calculated. Circular dichroism and fluorescence spectroscopic results corroborate the DSC observations and indicate a protein conformation with poorly defined tertiary structure and high content of secondary structure in the presence of 50 mM 4-chlorobutan-1-ol at a temperature corresponding to the second transition. Similar results have been observed at pH 3.9., (Copyright 2003 Elsevier B.V.)

Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 m trehalose was observed to raise the transition temperature, Tm of RNase A by as much as 18 degrees C and Gibbs free energy by 4.8 kcal mol-1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (DeltaCp) in trehalose solutions for all the studied proteins. An increase in the DeltaG and a decrease in the DeltaCp values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (DeltaG versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 m concentration 4-7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.

Rat eosinophils contain eosinophil-associated ribonucleases (Ears) in their granules. Ears are thought to be synthesized as pre-forms and stored in the granules as mature forms. However, the N-terminal amino acid of mature Ear-1 and Ear-2 is still controversial. Therefore, we prepared two recombinant mature forms of Ear-1 and Ear-2 in which the N-terminal amino acids are Ser24 (S) [Ear-1 (S) and Ear-2 (S)] and Gln26 (Q) [Ear-1 (Q) and Ear-2 (Q)], and analyzed their biological activities by comparing them with those of pre-form Ear-1 and pre-form Ear-2. The four mature Ears showed RNase A activity as well as bovine pancreatic RNase A activity, but pre-Ear-1 and pre-Ear-2 showed no RNase A activity. Mature Ear-1 (Q) and mature Ear-2 (Q) showed more potent RNase A activity than mature Ear-1 (S) and mature Ear-2 (S), respectively. The RNase A activities of mature Ear-1 (Q) and mature Ear-2 (Q) were reduced by treatment at 96 degrees C for 20 min or with RNase inhibitor. The growth of Escherichia coli was inhibited by both pre-Ears and mature Ears in a concentration-dependent manner, and was almost completely suppressed at 1.0 microM. The bactericidal activities of mature Ear-1 (Q) and mature Ear-2 (Q) were not inhibited by RNase inhibitor, but was increased by treatment at 96 degrees C for 20 min.

Arginine is one of the universal reagents that are effective in assisting refolding of recombinant proteins from inclusion bodies. The mechanism of the effects of arginine on refolding has remained, however, to be elucidated. Here we show that arginine does not stabilize proteins against heat treatment, as demonstrated by little change in melting temperature. It does increase reversibility of thermal melting and reduce aggregation under thermal stress. The observations suggest that arginine may not facilitate refolding, but may suppress aggregation of the proteins during refolding.

The kinetics of the reversible thermal unfolding, irreversible thermal unfolding, and reductive unfolding processes of bovine pancreatic ribonuclease A (RNase A) were investigated in NaCl/Pi solutions. Image parameters including Shannon entropy, Hamming distance, mutual information and correlation coefficient were used in the analysis of the CD and 1D NMR spectra. The irreversible thermal unfolding transition of RNase A was not a cooperative process, pretransitional structure changes occur before the main thermal denaturation. Different dithiothreitol (dithiothreitolred) concentration dependencies were observed between 303 and 313 K during denaturation induced by a small amount of reductive reagent. The protein selectively follows a major unfolding kinetics pathway with the selectivity can be altered by temperature and reductive reagent concentration. Two possible explanations of the selectivity mechanism were discussed.

With the aim to distinguish between local and global conformational changes induced by trifluoroethanol in RNase A, spectroscopic and activity measurements in combination with proteolysis by unspecific proteases have been exploited for probing structural transitions of RNase A as a function of trifluoroethanol concentration. At > 30% (v/v) trifluoroethanol (pH 8.0; 25 degrees C), circular dichroism and fluorescence spectroscopy indicate a cooperative collapse of the tertiary structure of RNase A coinciding with the loss of its enzymatic activity. In contrast to the denaturation by guanidine hydrochloride, urea or temperature, the breakdown of the tertiary structure in trifluoroethanol is accompanied by an induction of secondary structure as detected by far-UV circular dichroism spectroscopy. Proteolysis with the nonspecific proteases subtilisin Carlsberg or proteinase K, both of which attack native RNase A at the Ala20-Ser21 peptide bond, yields refined information on conformational changes, particularly in the pretransition region. While trifluoroethanol at concentrations > 40% results in a strong increase of the rate of proteolysis and new primary cleavage sites (Tyr76-Ser77, Met79-Ser80) were identified, the rate of proteolysis at trifluoroethanol concentrations < 40% (v/v) is much smaller (up to two orders of magnitude) than that of the native RNase A. The proteolysis data point to a decreased flexibility in the surrounding of the Ala20-Ser21 peptide bond, which we attribute to subtle conformational changes of the ribonuclease A molecule. These changes, however, are too marginal to alter the overall catalytic and spectroscopic properties of ribonuclease A.

Ribonuclease-A (RNase-A) has been a model for studying protein folding and unfolding. However, we show here that its unfolding at neutral pH is complicated by aggregation. Circular dichroism thermal scans showed that reversibility of RNase-A after heating is only about 63%. In accordance with this observation, native-polyacrylamide gel electrophoresis of the sample heated at 75 degrees C showed formation of soluble oligomers. Ammonium sulfate at 0.4 M caused about a 3 degrees C higher melting temperature and nearly complete reversibility, while glycine and NaCl at 0.4 M significantly increased reversibility and decreased aggregation without affecting melting temperature. These results demonstrate that aggregation makes thermal unfolding of RNase-A at least partially irreversible and salts and glycine increase reversibility and decrease aggregation.

Purpose: Angiogenin is a potent positive mediator of neovascularization, a process required for both primary tumor growth and metastasis. In the present study, the effect of a fully phosphorothioated antisense oligodeoxynucleotide, designated JF2S, targeting the AUG translation initiation codon region of human angiogenin, on human prostate tumor development and metastasis in athymic mice was examined., Experimental Design: JF2S was evaluated for its capacity to affect in vitro synthesis of angiogenin and subsequent tumorigenicity of transiently transfected prostate tumor cells in mice. In vivo treatment experiments were then conducted in which JF2S was used to prevent formation of tumors in an ectopic model and metastasis in an orthotopic model., Results: Transient transfection of tumor cells with JF2S inhibited both angiogenin gene expression in vitro and tumorigenicity of these transfected cells in athymic mice. In therapy experiments, local treatment with JF2S completely protected mice from developing prostate tumors after s.c. injection of PC-3 human prostate tumor cells (P < 0.0001, survivor analysis). Most importantly, systemic prophylactic administration of JF2S prevented, in 47% of mice, formation of regional iliac lymph node micrometastases arising from primary tumors growing in the more natural orthotopic prostate setting (P = 0.0003, Fisher's exact test). Furthermore, total protection from regional metastasis occurred in those mice in which JF2S treatment successfully diminished human angiogenin expression in vivo. Tumor-associated angiogenesis was also impaired by JF2S treatment. When therapy was delayed until all of the mice harbored primary tumors in the prostate, the incidence of regional metastasis was still significantly decreased (P < 0.005, survivor analysis)., Conclusions: These findings demonstrate that human prostate cancer establishment and spread in athymic mice is extremely susceptible to targeted disruption of tumor-derived human angiogenin gene expression. Therefore, angiogenin is a valid target against which to devise preventative strategies for prostate cancer metastasis.

Proteins exposed to oxidative stress are degraded via proteolytic pathways. In the present study, we undertook a series of in vitro experiments to establish a correlation between the structural changes induced by mild oxidation of the model protein RNase A and the proteolytic rate found upon exposure of the modified protein toward the isolated 20 S proteasome. Fourier transform infrared spectroscopy was used as a structure-sensitive probe. We report here strong experimental evidence for oxidation-induced conformational rearrangements of the model protein RNase A and, at the same time, for covalent modifications of amino acid side chains. Oxidation-related conformational changes, induced by H(2)O(2) exposure of the protein may be monitored in the amide I region, which is sensitive to changes in protein secondary structure. A comparison of the time- and H(2)O(2) concentration-dependent changes in the amide I region demonstrates a high degree of similarity to spectral alterations typical for temperature-induced unfolding of RNase A. In addition, spectral parameters of amino acid side chain marker bands (Tyr, Asp) revealed evidence for covalent modifications. Proteasome digestion measurements on oxidized RNase A revealed a specific time and H(2)O(2) concentration dependence; at low initial concentration of the oxidant, the RNase A turnover rate increases with incubation time and concentration. Based on these experimental findings, a correlation between structural alterations detected upon RNase A oxidation and proteolytic rates of RNase A is established, and possible mechanisms of the proteasome recognition process of oxidatively damaged proteins are discussed.

RNase A and its minor and major dimers were digested with subtilisin under controlled conditions. The major dimer was found to be slightly more resistant, the minor dimer markedly less resistant to subtilisin than monomeric RNase A. Two S-proteins formed for each RNase A species, one starting with Ser-21, the other with Ser-22. Their relative proportions indicate that the structure of the minor dimer, whose identity with that of a RNase A dimer shown to be 3D domain-swapped is strongly suggested by recent work [S. Sorrentino et al. (2000) FEBS Lett. 466, 35-39], makes its peptide bond between Ser-21 and Ser-22 more accessible to subtilisin than it is in RNase A and its major dimer. Moreover, (i) both subunits constituting the minor dimer are more susceptible to subtilisin than monomeric RNase A, and (ii) the susceptible bonds in one of its two exchanging N-terminal arms are more accessible to the protease than in the other. The properties of the major dimer suggest that its structure could be different.

The DeltaG degrees (N)(-)(D) value obtained from extrapolation to zero denaturant concentration by the linear extrapolation method (LEM) is commonly interpreted to represent the Gibbs energy difference between native (N) and denatured (D) ensembles at the limit of zero denaturant concentration. For DeltaG degrees (N)(-)(D) to be interpreted solely in terms of N and D, as is common practice, it must be shown to be independent of denaturant concentration. Because DeltaG degrees (N)(-)(D) is often observed to be dependent on the nature of the denaturant, it is necessary to determine the circumstances under which DeltaG degrees (N)(-)(D) can be interpreted as a property solely of the protein. Here, we use proton inventory, a thermodynamic property of both the native and denatured ensembles, to monitor the thermodynamic character of denaturant-dependent aspects of N and D ensembles and the N right arrow over left arrow D transition. Use of a thermodynamic rather than a spectral parameter to monitor denaturation provides insight into the manner in which denaturant affects the meaning of DeltaG degrees (N)(-)(D) and the nature of the N right arrow over left arrow D transition. Three classes of proteins are defined in terms of the thermodynamic behaviors of their N right arrow over left arrow D transition and N and D ensembles. With guanidine hydrochloride as a denaturant, the classification of protein denaturations by these procedures determines when the LEM gives readily interpretable DeltaG degrees (N)(-)(D) values with this denaturant and when it does not.

The folding of ribonuclease A (RNase A) has been extensively studied by characterizing the disulfide containing intermediates using different experimental conditions and analytical techniques. So far, some aspects still remain unclear such as the role of the loop 65-72 in the folding pathway. We have studied the oxidative folding of a RNase A derivative containing at position 67 the substitution Asn --> isoAsp where the local structure of the loop 65-72 has been modified keeping intact the C65-C72 disulfide bond. By comparing the folding behavior of this mutant to that of the wild-type protein, we found that the deamidation significantly decreases the folding rate and alters the folding pathway of RNase A. Results presented here shed light on the role of the 65-72 region in the folding process of RNase A and also clarifies the effect of the deamidation on the folding/unfolding processes. On a more general ground, this study represents the first characterization of the intermediates produced along the folding of a deamidated protein.

31P NMR spectroscopy has been used to show that the activity of RNase A, which is lowered in the presence of urea, can be recovered with trimethylamine-N-oxide (TMAO). A 1:1 ratio of TMAO:urea was sufficient to recover the enzyme activity. (1)H nuclear Overhauser effect spectroscopy NMR studies with RNase A have shown that even at relatively low effective concentrations of TMAO, some modification of the three-dimensional structure of the biomolecule is apparent.

The role of protein disulfide isomerase (PDI) in the regeneration of ribonuclease A with dithiothreitol (DTT) was investigated at three different temperatures. The rates of formation of the native protein were markedly increased in the presence of PDI, 9-fold at 15 degrees C, 6-fold at 25 degrees C and 62-fold at 37 degrees C, respectively. In the presence of PDI, major changes were found in the distribution of intermediates in the three-disulfide region at 25 and 15 degrees C and also in the one-disulfide region at 15 degrees C, with the fast accumulation of the two native-like species des-[65-72] and des-[40-95]. The present results indicate that PDI does not alter the two major parallel pathways involving des-[65-72] and des-[40-95] in the regeneration of ribonuclease A with DTT.

Reactive aldehydes derived from reducing sugars and peroxidation of lipids covalently modify proteins and may contribute to oxidative tissue damage. We recently described another mechanism for generating reactive aldehydes from free alpha-amino acids. The pathway begins with myeloperoxidase, a heme enzyme secreted by activated neutrophils. Conversion of alpha-amino acids to aldehydes requires hypochlorous acid (HOCl), formed from H2O2 and chloride by myeloperoxidase. When L-serine is the substrate, HOCl generates high yields of glycolaldehyde. We now demonstrate that a model protein, ribonuclease A (RNase A), exposed to free L-serine and HOCl exhibits the biochemical hallmarks of advanced glycation end (AGE) products -- browning, increased fluorescence, and cross-linking. Furthermore, Nepsilon-(carboxymethyl)lysine (CML), a chemically well-characterized AGE product, was generated on RNase A when it was exposed to reagent HOCl-serine, the myeloperoxidase-H2O2-chloride system plus L-serine, or activated human neutrophils plus L-serine. CML production by neutrophils was inhibited by the H2O2 scavenger catalase and the heme poison azide, implicating myeloperoxidase in the cell-mediated reaction. CML was also generated on RNase A by a myeloperoxidase-dependent pathway when neutrophils were activated in a mixture of amino acids. Under these conditions, we observed both L-serine-dependent and L-serine-independent pathways of CML formation. The in vivo production of glycolaldehyde and other reactive aldehydes by myeloperoxidase may thus play an important pathogenic role by generating AGE products and damaging tissues at sites of inflammation.

2,2,2-Trifluoroethanol (TFE) is known to stabilize peptide helices by strengthening hydrogen bonds. On the other hand, TFE destabilizes native proteins, as we confirm here, presumably by weakening the hydrophobic interaction. The stability of the pH 4 folding intermediate of apomyoglobin is known to depend both on the strength of the individual A, G, and H helices and on hydrophobic interactions between helices. We ask which effect of TFE dominates in this case: strengthening helices or weakening hydrophobic interactions between helices? Protein stability is measured by denaturant-induced unfolding curves, and two-state unfolding is tested by monitoring both far-UV CD and tryptophan fluorescence emission. Low concentrations of TFE strongly stabilize the pH 4 folding intermediate. Moreover, low concentrations of TFE compensate for helix-destabilizing mutations in the A and G helices. Consequently, enhancing helix propensity, rather than weakening the hydrophobic interaction, is the dominant effect of TFE on the folding intermediate. This result agrees with earlier mutational evidence that helix propensities are very important in determining the stability of the pH 4 intermediate. Although TFE destabilizes native holomyoglobin, as well as native lysozyme and ribonuclease A, nevertheless, TFE stabilizes native apomyoglobin.

We have investigated the effect of two simple sugars, glucose and sucrose, on the extraction of ribonuclease A by AOT-isooctane reversed micelles. Including the sugars at concentrations up to 0.75 M in the feed solution resulted in moderate improvements in the forward transfer efficiency. The greatest effects were seen observed in the backward transfer step where both the protein recovery yield and the activity of the protein were greatly increased. Protein transfer and activity yields were also dependent on the AOT concentration. We suggest that the presence of sucrose, which was solubilized into the reversed micelles, results in preferential hydration of ribonuclease A, reducing the protein-surfactant interactions., (Copyright 1998 John Wiley & Sons, Inc.)

Syncephapepsin is a fungal aspartic proteinase from Syncephalastrum racemosum. By using the property of syncephapepsin after having increased activity at higher temperature, two rapid purification protocols were developed. In the former case, a crude extract was initially diluted fivefold with an activity assay buffer and heated at 50 degrees C overnight. In this situation, syncephapepsin would digest most of the proteins that the crude extract contained. Subsequently, syncephapepsin of the crude extract was precipitated from 50 to 70% of ammonium sulfate and the preparation was then directly applied to the Superdex 200 HR FPLC column. In this manner, syncephapepsin was rapidly purified to apparent homogeneity within 24 h. In this report, an alternative method of purification is also provided. Compared with the procedure mentioned above, the heating step was proceeded after FPLC chromatography through which the same result was obtained. Using cytochrome c and RNase A as substrates, the cleavage sites of both substrates were identified by HPLC peptide mapping. The results showed that syncephapepsin had a broad specificity. Residues recognized to be cleaved were primarily those of trypsin and chymotrypsin and Lys was the most susceptible., (Copyright 1998 Academic Press.)

Urea in renal medullas is sufficiently high to perturb macromolecules, yet the cells survive and function. The counteracting osmolytes hypothesis holds that methylamines, such as glycine betaine (betaine) and glycerophosphocholine (GPC) in renal medullas, stabilize macromolecules and oppose the effects of urea. Although betaine counteracts effects of urea on macromolecules in vitro and protects renal cells from urea in tissue culture, renal cells accumulate GPC rather than betaine in response to high urea both in vivo and in tissue culture. A proposed explanation is that GPC counteracts urea more effectively than betaine. However, we previously found GPC slightly less effective than betaine in counteracting inhibition of pyruvate kinase activity by urea. To test another macromolecule, we now compare GPC and betaine in counteracting reduction of the thermal stability of Rnase A by urea. We find that urea decreases the thermal transition temperature and that betaine and GPC increase it, counteracting urea approximately equally. Therefore, the preference for GPC in response to high urea presumably has some other basis, such as a lower metabolic cost of GPC accumulation.

It was previously revealed [Yamaguchi, H. and Uchida, M. (1996) J. Biochem. 120, 474-477] that both intra- and extramolecular high-mannose type Asn-glycans promote the renaturation of reductively denatured bovine pancreatic RNases A and B under oxidation conditions. To characterize the conformational changes of the polypeptides during the renaturation promoted by the intramolecular Asn-glycans, RNase B was compared with its nonglycosylated form, RNase A, as to the features of the regeneration from their reductively denatured species under Cu2+-catalyzed oxidation conditions. The refolding intermediates of RNase B, as compared with those of RNase A, seemed to contain much less impaired disulfide linkages. In agreement with this finding, the proper refolding of RNase B was much faster than that of RNase A, as revealed by the intrinsic fluorescence and 1-anilino-8-naphthalenesulfonate binding of the refolding intermediates. Such a promoting effect was also observed for extramolecular Asn-glycans of the complex as well as of the high-mannose type. In contrast, common mono-, oligo-, and polysaccharides, but not yeast mannan, exhibited much lower stimulatory effects on the oxidative refolding of RNase A.

Smokers have a significantly higher risk for developing coronary and cerebrovascular disease than nonsmokers. Advanced glycation end products (AGEs) are reactive, cross-linking moieties that form from the reaction of reducing sugars and the amino groups of proteins, lipids, and nucleic acids. AGEs circulate in high concentrations in the plasma of patients with diabetes or renal insufficiency and have been linked to the accelerated vasculopathy seen in patients with these diseases. Because the curing of tobacco takes place under conditions that could lead to the formation of glycation products, we examined whether tobacco and tobacco smoke could generate these reactive species that would increase AGE formation in vivo. Our findings show that reactive glycation products are present in aqueous extracts of tobacco and in tobacco smoke in a form that can rapidly react with proteins to form AGEs. This reaction can be inhibited by aminoguanidine, a known inhibitor of AGE formation. We have named these glycation products "glycotoxins." Like other known reducing sugars and reactive glycation products, glycotoxins form smoke, react with protein, exhibit a specific fluorescence when cross-linked to proteins, and are mutagenic. Glycotoxins are transferred to the serum proteins of human smokers. AGE-apolipoprotein B and serum AGE levels in cigarette smokers were significantly higher than those in nonsmokers. These results suggest that increased glycotoxin exposure may contribute to the increased incidence of atherosclerosis and high prevalence of cancer in smokers.

The effect of 2,2,2-trifluoroethanol (TFE) on the structure of an all beta-sheet protein, cardiotoxin analogue 111 (CTX III) from the Taiwan cobra (Naja naja atra) is studied. It is found that high concentrations (> 80% v/v) of TFE induced a beta-sheet to alpha-helix structural transition. It is found that in denatured and reduced CTX III (rCTX III) helical conformation is induced even upon addition of low concentrations (> 10% v/v) of TFE. Using three other proteins, namely, ribonuclease A (RNase A), lysozyme and alpha-lactalbumin, it is been observed that helix-induction by TFE is intricately linked to drastic destabilization of native tertiary structural interactions in the proteins.

Nonenzymatic glycation of proteins by glucose leading to the formation of toxic and immunogenic advanced glycation end products (AGEs) may be a major contributor to the pathological manifestations of diabetes mellitus, aging, and, possibly, neurodegenerative diseases such as Alzheimer's. We tested the in vitro inhibition of antigenic AGE formation on bovine serum albumin, ribonuclease A, and human hemoglobin by various vitamin B1 and B6 derivatives. Among the inhibitors, pyridoxamine and thiamine pyrophosphate potently inhibited AGE formation and were more effective than aminoguanidine, suggesting that these two compounds may have novel therapeutic potential in preventing vascular complications of diabetes. An unexpected finding was that aminoguanidine inhibited the late kinetic stages of glycation much more weakly than the early phase.

Four single mutants (P42A, P93A, P114A, and P117A) of bovine pancreatic ribonuclease A (RNase A) in which each mutant has one of the four prolines of RNase A changed to alanine were prepared. The physical properties of these four mutants indicate that their native structure is essentially identical to that of wild-type RNase A. The disulfide-intact forms of these proteins were denatured in guanidine hydrochloride (Gdn.HCl) and then refolded by dilution of the Gdn.HCl. Single-jump folding, single-jump unfolding, and double-jump unfolding/folding stopped-flow experiments were carried out on wild-type and the four proline mutants of RNase A using absorption detection to follow the folding kinetics. The single-jump folding experiments carried out at six different final Gdn.HCl concentrations indicate that the folding rate constants of individual steps for the mutants are similar to those of wild-type RNase A. The Tyr92-Pro93 peptide bond has a cis conformation in native wild-type RNase A, and the results from our double-jump stopped-flow experiments indicate that the Tyr92-Ala93 peptide bond in the P93A mutant of RNase A is also cis in the native state. The existence of two cis peptide bonds (preceding Pro93 and Pro114) in wild-type RNase A is probably due to (as-yet-unidentified) long-range interactions, and such interactions are presumably the origin of a cis peptide bond even when alanine is substituted for Pro93. The data from the double-jump stopped-flow experiments are interpreted in terms of a folding/unfolding model. This model specifies the cis/trans isomerization state of the unfolded species (Uvf, Uf, Um, and Us) at each X-Pro peptide bond. Also, this model confirms the existence of several previously postulated chain-folding initiation sites.

Osmolytes are small organic solutes accumulated intracellularly by many organisms as they adapt to environmental stresses. Compatible osmolytes, a functional class of osmolytes, increase protein stability while having little or no effect on protein function. To investigate the interrelationships between protein stability, function, and internal dynamics, a hydrogen exchange (HX) quench method was established and used to study the effects of sucrose (a typical compatible osmolyte) on the structural fluctuations of ribonuclease A. It was found that the HX rates of the amide protons with intermediate rates are not affected by 1 M sucrose, but the slow-exchanging amide protons exchange even slower in 1 M sucrose. The protection factors of the slow-exchanging protons fall into a comparatively narrow range while those of the intermediate-exchanging protons vary widely. In agreement with the two-process model [Woodward, C.K., & Hilton, B. D. (1980) Biophys. J. 32, 561-575], we conclude that for those slow-exchanging amide protons, the exchange occurs mainly from the compact unfolded state ensemble of the protein. The internal dynamics leading to slow exchange involve exposure of large protein surface areas, similar to that which occurs upon the unfolding of protein. Because sucrose opposes such an increase in protein surface area exposure, both the slow HX rates and the protein stability are affected by sucrose. For those amide protons with fast and intermediate HX rates, the exchange occurs mainly from the native state ensemble of the protein. The internal dynamics involved in the exchange are localized without much surface area change, and functionally important structural fluctuations are likely to occur within this dynamic range.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes the oxidation of protein sulfhydryl groups and the isomerization and reduction of protein disulfide bonds. Saccharomyces cerevisiae cells lacking PDI are inviable. PDI is a component of many different protein processing complexes, and the actual activity of PDI that is required for cell viability is unclear. A cDNA that codes for rat PDI fused to the alpha-factor pre-pro segment was expressed in a protease-deficient strain of S. cerevisiae under the control of an ADH2-GAPDH hybrid promoter. The cells processed the resulting protein and secreted it into the medium as a monomer, despite having a KDEL or HDEL sequence at its C-terminus. The typical yield of isolated protein was 2 mg per liter of culture. The catalytic activity of the PDI from S. cerevisiae was indistinguishable from that of PDI isolated from bovine liver. This expression system is unique in allowing the same plasmid to be used both to complement pdi1 delta S. cerevisiae and to produce PDI for detailed in vitro analyses. Correlations of the in vivo behavior and in vitro properties of PDI are likely to reveal structure-function relationships of biological importance.

Inactivation of pancreatic RNAase A occurs in guanidinium chloride (GdmCl) at low concentrations before the unfolding of the molecule as a whole can be detected [Liu and Tsou (1987) Biochim. Biophys. Acta 916, 455-464]. We have now shown that the rate of digestion of the RNAase molecule by either trypsin or proteinase K increases significantly at low concentrations of GdmCl where the enzyme is largely inactivated, but fluorescence and absorption measurements reveal no conformational changes. N-Terminal sequence analysis of the peptide fragments generated shows that proteolysis occurs primarily at or near the active site. The decrease in activity of RNAase at low concentrations of GdmCl is therefore due to partial unfolding of the molecule, particularly at the active site and not to an inhibition by the denaturant.

The exposure of ribonuclease A to trichloroacetic acid was earlier shown to alter the conformation of the protein resulting in reduced enzymatic activity (Sagar, A. J., Subbiah, V., and Pandit, M. W. (1989) Biochim. Biophys. Acta 995, 144-150). We have studied the structure and enzymatic activity of ribonuclease A treated with trichloroacetic acid over a wide range of acid concentrations (0-40%). The far ultraviolet circular dichroism spectra of ribonuclease A, on exposure to acid concentrations less than 10%, indicated an exceptionally high degree of chiral structure. Exposure of ribonuclease A to acid concentrations between 10 and 30% resulted in the formation of a molecule with significant chiral structure (conventionally assigned to residual secondary structure) but reduced tertiary structure (characteristics very similar to those of molten globule). Increased binding of the hydrophobic probe 1-anilinonaphthalene-8-sulfonate to the enzyme treated with 15-30% acid, as compared with the untreated or completely unfolded protein, supported the existence of a state having characteristics of molten globule. Reversed phase high performance liquid chromatography corroborated the data obtained by circular dichroism as well as 1-anilino-naphthalene-8-sulfonate-binding studies. Beyond acid concentrations of 30%, the ribonuclease is completely denatured. The trichloroacetic acid-induced unfolding is shown to be completely reversible.

Guanidinium chloride is a commonly used denaturant to unfold native proteins and to determine their Gibbs free energy of stabilization, delta Gstab. Here we show that this denaturant has a dual role for the stability and the folding of the model protein ribonuclease T1. When present at low concentration (0-0.3 M), guanidinium chloride stabilizes the folded protein toward thermal and urea-induced unfolding and decreases the rate of unfolding. At high concentration the function of guanidinium chloride as a denaturant dominates and ribonuclease T1 is cooperatively unfolded. Ribonuclease T1 is also strongly stabilized by other salts, such as NaCl, at low concentrations, and the dependence of the thermal stability on salt concentration is not linear. Such a complex behavior was not found in control experiments with pancreatic ribonuclease A. The stabilization in the presence of low concentrations of guanidinium chloride originates probably from the binding of guanidinium ions to one or a few cation binding sites that exist in native ribonuclease T1. It is not observed when an additional salt, NaCl, is present simultaneously. The favorable interaction of guanidinium chloride with the native protein leads to increased values for delta Gstab, when unfolding transitions induced by guanidinium chloride are analyzed on the basis of the two-state model by the linear extrapolation procedure. The noncoincidence of these delta Gstab values with stability data derived from urea-induced or thermal unfolding transitions does not imply that the two-state model is not appropriate but that the linear extrapolation to zero molar denaturant is incorrect.(ABSTRACT TRUNCATED AT 250 WORDS)

The guanidinium chloride (GdmCl) denaturation of RNAase A, lysozyme and metmyoglobin was investigated at several pH values by using absorbance measurements at 287, 300 and 409 nm respectively. From these measurements the free-energy change on denaturation, delta Gapp., was calculated, assuming a two-state mechanism, and values of delta Gapp. at zero concentration of the denaturant were measured. For each protein all delta Gapp. values were adjusted to pH 7.00 by using the appropriate relationship between delta Gapp. and pH. Dependence of the adjusted delta Gapp. value on GdmCl concentration increases for metmyoglobin and decreases for the other two proteins as the denaturant concentration decreases. It has been shown that these are expected results if the presence of the acid-denatured state during the GdmCl denaturation of proteins is considered.

The stability of a freeze-dried model protein, ribonuclease A (RNase), was investigated under accelerated storage conditions at 45 degrees C for time periods up to 60 days. Because RNase is a fairly stable molecule around pH 7, lyophilization was performed in phosphate buffers at pH 4.0 or 10.0 to accelerate degradation kinetics. Degradation was studied by measuring enzymatic activity, the concentrations of soluble monomeric RNase, soluble aggregated (polymerized) RNase, and insoluble aggregated RNase following reconstitution of the lyophilized material at different times. The presence of air in the vial headspace accelerated degradation in the solid state in all cases. When argon or nitrogen was employed in the headspace, degradation kinetics were reduced, implying that molecular oxygen was involved in the degradation process. This interpretation was supported by the observation that 0.05% (w/v) EDTA in the formulation prior to freeze-drying retarded RNase degradation dramatically. EDTA was believed to chelate cations which may have been introduced with the buffer salts in trace quantities sufficient to catalyze autoxidation reactions. Incorporation of antioxidants ascorbic acid (at pH 4.0) and POBN (a spin trap which could have functioned as an antioxidant at pH 10.0) accelerated the degradation of RNase and appeared, in both cases, to be involved in interactions with the protein molecules. Additionally, in the presence of the antioxidants RNase degradation appeared to be accelerated by light. Although there is strong support for the oxidative hypothesis, the possibility of other competing reactions cannot be discounted. These investigations demonstrate the importance of challenging the extrapolation of some of our well-established ideas concerning small molecule solution kinetics to macromolecules in the solid state.