Biomedical subjects

Mahesh Narayan

Publications and source records attributed to Mahesh Narayan.

16 recordsLinked to original sources

PubMed2006

Analysis and comparison of active constituents in commercial standardized silymarin extracts by liquid chromatography-electrospray ionization mass spectrometry.

A sensitive method for the simultaneous quantitation of six active constituents in commercial silymarin standardized extracts was developed based on liquid chromatography (LC) in combination with mass spectrometry (MS). The six main active constituents, namely, silydianin, silychristin, diastereoisomers of silybin (silybin A and B), and diastereoisomers of isosilybin (isosilybin A and B) were completely separated and quantified by LC/MS. Silymarin obtained from Sigma-Aldrich Co. was evaluated and used as standard reference material for the six individual constituents in comparing the relative content of silymarin and the relative ratio of each constituent in commercial standardized silymarin extracts, respectively. Significant variation was found between different commercial silymarin sources. As a result, this method has proven useful in evaluating and quantifying the six active constituents in commercial milk thistle extracts. The calibration curves were over the range from 0.25 to 100 microg/mL for silychristin and silydianin, and from 0.10 to 100 microg/mL for silybin A, silybin B, isosilybin A and isosilybin B, respectively (r(2)> or =0.9958). For all six active constituents, the overall intra-day precision values, based on the relative standard deviation replicate for four QC levels, ranged from 1.18% to 12.4% and accuracy ranged from 89.4% to 112%. This methodology could easily be incorporated into standardized testing to assess content uniformity including lot-to-lot variation as part of routine process controls as well as a means to describe cross-product variation among the exiting marketed formulations.

Chromatography, Liquid↗

PubMed2006

A localized specific interaction alters the unfolding pathways of structural homologues.

Reductive unfolding studies of proteins are designed to provide information about intramolecular interactions that govern the formation (and stabilization) of the native state and about folding/unfolding pathways. By mutating Tyr92 to G, A, or L in the model protein, bovine pancreatic ribonuclease A, and through analysis of temperature factors and molecular dynamics simulations of the crystal structures of these mutants, it is demonstrated that the markedly different reductive unfolding rates and pathways of ribonuclease A and its structural homologue onconase can be attributed to a single, localized, ring-stacking interaction between Tyr92 and Pro93 in the bovine variant. The fortuitous location of this specific stabilizing interaction in a disulfide-bond-containing loop region of ribonuclease A results in the localized modulation of protein dynamics that, in turn, enhances the susceptibility of the disulfide bond to reduction leading to an alteration in the reductive unfolding behavior of the homologues. These results have important implications for folding studies involving topological determinants to obtain folding/unfolding rates and pathways, for protein structure-function prediction through fold recognition, and for predicting proteolytic cleavage sites.

Animals↗

PubMed2005

The oxidative folding rate of bovine pancreatic ribonuclease is enhanced by a covalently attached oligosaccharide.

Bovine pancreatic ribonuclease B (RNase B) differs from RNase A by the presence of an oligosaccharide moiety covalently attached to Asn 34. Oxidative folding studies of RNase B were carried out at different temperatures using DTT(ox) as the oxidizing agent, and the results were compared with those for RNase A. The oxidative folding rates of RNase B are between 1.7 and 1.3 times faster than those of RNase A at the temperatures that were investigated. The folding pathways of RNase B were determined to be similar to those of RNase A in that two structured intermediates, each lacking one native disulfide bond, were found to populate the regeneration pathways at 25 degrees C and pH 8.3. The thermodynamic stabilities of these two glycosylated intermediates, and their rates of formation from their unstructured precursors in the rate-determining step, were found to be higher than those of their unglycosylated counterparts from RNase A. Thus, the underlying cause for the faster rate of oxidative regeneration of native RNase B appears to be both thermodynamic and kinetic due to the higher stability, and faster rate of formation, of the intermediates of RNase B compared to those of RNase A.

Animals↗

PubMed2005

Drug utilization in the pediatric intensive care unit: monitoring prescribing trends and establishing prioritization of pharmacotherapeutic evaluation of critically ill children.

The primary objective of this study was to characterize the drug exposure for children hospitalized in the authors' institution's pediatric intensive care unit for the year 2002. Secondary objectives included the examination of drug utilization differences among various age criteria and the suitability of the most prevalent resources for pediatric dosing guidance. Many of the most commonly prescribed agents in the pediatric intensive care unit fall into the broad categories of pain management/sedation and anti-infectives. Based on the generally narrow windows afforded by each of these drug classes, it is obvious that more, well-defined investigations in critically ill children are warranted. The existing dosing guidance for many of these agents is neither generalizable nor sufficient to accommodate the diversity in pediatric intensive care unit patients, and the current drug monographs fall short of any practical dosing information.

Adolescent↗

PubMed2004

Trimethylamine-N-oxide modulates the reductive unfolding of onconase.

The physiological osmolyte trimethylamine-N-oxide (TMAO) stabilizes proteins by decreasing the entropy of the unfolded state through a solvophobic effect. Our studies on the effect of TMAO on the reductive unfolding of onconase (ONC) to form its reductive intermediate, des [30-75], indicate that TMAO diminishes the reductive unfolding rate of the protein although it does not significantly affect the stability of the native protein relative to its denatured state. Since the reductive unfolding of ONC is a local event, our studies provide direct evidence for a TMAO-induced local structural change that reduces the rate of redox-dependent protein unfolding. The implications of our findings for protein folding/unfolding are discussed.

Enzyme Inhibitors↗

PubMed2004

Dissimilarity in the reductive unfolding pathways of two ribonuclease homologues.

Using DTT(red) as the reducing agent, the kinetics of the reductive unfolding of onconase, a frog ribonuclease, has been examined. An intermediate containing three disulfides, Ir, that is formed rapidly in the reductive pathway, is more resistant to further reduction than the parent molecule, indicating that the remaining disulfides in onconase are less accessible to DTT(red). Disulfide-bond mapping of Ir indicated that it is a single species lacking the (30-75) disulfide bond. The reductive unfolding pattern of onconase is consistent with an analysis of the exposed surface area of the cysteine sulfur atoms in the (30-75) disulfide bond, which reveals that these atoms are about four- and sevenfold, respectively, more exposed than those in the next two maximally exposed disulfides. By contrast, in the reductive unfolding of the homologue, RNase A, there are two intermediates, arising from the reduction of the (40-95) and (65-72) disulfide bonds, which takes place in parallel, and on a much longer time-scale, compared to the initial reduction of onconase; this behavior is consistent with the almost equally exposed surface areas of the cysteine sulfur atoms that form the (40-95) and (65-72) disulfide bonds in RNase A and the fourfold more exposed cysteine sulfur atoms of the (30-75) disulfide bond in onconase. Analysis and in silico mutation of the residues around the (40-95) disulfide bond in RNase A, which is analogous to the (30-75) disulfide bond of onconase, reveal that the side-chain of tyrosine 92 of RNase A, a highly conserved residue among mammalian pancreatic ribonucleases, lies atop the (40-95) disulfide bond, resulting in a shielding of the corresponding sulfur atoms from the solvent; such burial of the (30-75) sulfur atoms is absent from onconase, due to the replacement of Tyr92 by Arg73, which is situated away from the (30-75) disulfide bond and into the solvent, resulting in the large exposed surface-area of the cysteine sulfur atoms forming this bond. Removal of Tyr92 from RNase A resulted in the relatively rapid reduction of the mutant to form a single intermediate (des [40-95] Y92A), i.e. it resulted in an onconase-like reductive unfolding behavior. The reduction of the P93A mutant of RNase A proceeds through a single intermediate, the des [40-95] P93A species, as in onconase. Although mutation of Pro93 to Ala does not increase the exposed surface area of the (40-95) cysteine sulfur atoms, structural analysis of the mutant reveals that there is greater flexibility in the (40-95) disulfide bond compared to the (65-72) disulfide bond that may make the (40-95) disulfide bond much easier to expose, consistent with the reductive unfolding pathway and kinetics of P93A. Mutation of Tyr92 to Phe92 in RNase A has no effect on its reductive unfolding pathway, suggesting that the hydrogen bond between the hydroxyl group of Tyr92 and the carbonyl group of Lys37 has no impact on the local unfolding free energy required to expose the (40-95) disulfide bond. Thus, these data shed light on the differences between the reductive unfolding pathways of the two homologous proteins and provide a structural basis for the origin of this difference.

Animals↗

PubMed2004

Characterization of the fast-forming intermediate, des [30-75], in the reductive unfolding of onconase.

A fast-forming intermediate in the reductive unfolding of frog onconase (ONC), des [30-75], analogous to the des [40-95] intermediate found in the reductive unfolding of its structural homologue, bovine pancreatic ribonuclease A (RNase A), has been isolated and characterized. The midpoints of the thermal transition and chemical denaturing curves (representing global unfolding) indicate that the conformation of des [30-75] is considerably less stable than that of the parent molecule, suggesting that the (30-75) disulfide bond plays a significant role in the conformational stability of ONC. While des [30-75] is formed very quickly by a partial reduction of the parent molecule in a local unfolding step, it is not as easily susceptible to further reduction, indicating that its three disulfides are much more buried compared to the (30-75) disulfide bond in the parent protein. The nature of des [30-75] is similar to that of des [40-95] RNase A, in that des [30-75] ONC is also a disulfide-secure species. In addition, based on the resistance to mild reducing conditions, structured des species appear to form in ONC from unstructured three-disulfide-containing ensembles. This step is key in the oxidative folding of RNaseA, and is much faster in ONC than the formation of the structured des [40-95] species in RNase A.

Animals↗

PubMed2004

Simultaneous characterization of the reductive unfolding pathways of RNase B isoforms by top-down mass spectrometry.

A novel method for characterization of the simultaneous reductive unfolding pathways of five isoforms of bovine pancreatic ribonuclease B (RNase B) is demonstrated. The results indicate that each isoform unfolds reductively through two three-disulfide-containing structured intermediates before proceeding to the fully reduced form, as in the reductive unfolding pathways of the A variant lacking the carbohydrate chain. The rates of reduction of bovine pancreatic ribonuclease A (RNase A) and RNase B and the formation and consumption of their reductive intermediates are identical, indicating that the unfolding events necessary to expose disulfide bonds for reduction are not affected by the oligosaccharide. The method utilizes top-down mass spectrometry and a naturally occurring tag on the protein, viz. the carbohydrate moiety, to obtain unfolding information of an ensemble of protein isoforms and is a generally applicable methodological advance for conducting folding studies on mixtures of different proteins.

Animals↗

PubMed2004

Conformation-dependent interaction of alpha-lactalbumin with model and biological membranes: a spin-label ESR study.

Alpha-lactalbumin (alpha-LA) is biosynthesized and stored at the smooth endoplasmic reticulum (ER), then transferred to the Golgi lumen when prolactin stimulation of lactose biosynthesis and secretion takes place. Because both environments are composed of membranes, it was of interest to examine the interactions of alpha-LA with relevant model and biological membranes. Using the ESR spin-labeled fatty acid analog 5-doxyl stearic acid, we found evidence reflecting the insertion of "acid-shocked" molten globule (MG) alpha-LA into lecithin or phosphatidylserine (PS) multi-lamellar vesicles. An additional approximately 3 G immobilization was observed in the alpha-LA-lecithin sample versus the lipid alone. With PS, the increased immobilization was almost 6 G, reflecting an enhanced effect caused by strong electrostatic interactions between the positively charged protein with the negatively charged headgroup at pH 2.4. This was also reflected in the broadening of the PS:alpha-LA phase transition. Additionally, we have demonstrated that alpha-LA in its apo-form also shows similar insertion characteristics with both model and natural lipid membranes. Upon addition of calcium, the apo-form is released from the membrane as the Ca(2+)-bound protein.

Animals↗

PubMed2003

Assessing the magnitude of folding forces along the oxidative folding pathway of multi-disulfide-containing proteins.

Oxidative folding involves the acquisition of the native set of disulfide bonds and native structure of a protein from its reduced, unfolded form. Two major factors that influence the distribution of disulfide bonds (over their entropically favored probability of formation) during the regeneration, eventually favoring and sustaining the native set of disulfide bonds, are enthalpic interactions that lead to conformational ordering among intermediates and conformational folding, resulting in native (or nativelike) structure which protects the native disulfide bonds from reduction and intramolecular thiol-disulfide exchange reactions. Here, we report a novel technique to provide a quantitative description of the enthalpic and conformational folding forces present at any stage of the oxidative folding process of multi-disulfide-containing proteins which is made feasible by assessing their ability to form "structures" that are able to protect disulfide bonds from reduction.

Animals↗

PubMed2003

A novel method to determine thermal transition curves of disulfide-containing proteins and their structured folding intermediates.

The stability of a protein or of its folding intermediates is frequently characterized by its resistance to chemical and/or thermal denaturation. The folding/unfolding process is generally followed by spectroscopic methods such as absorbance, fluorescence, circular dichroism spectroscopy, etc. Here, we demonstrate a new method, by using HPLC, for determining the thermal unfolding transitions of disulfide-containing proteins and their structured folding intermediates. The thermal transitions of a model protein, ribonuclease A (RNase A), and a recently found unfolding intermediate of onconase (ONC), des [30-75], have been estimated by this method. Finally, the advantages of this method over traditional techniques are discussed by providing specific examples.

Chromatography, High Pressure Liquid↗

PubMed2003

Role of the [65-72] disulfide bond in oxidative folding of bovine pancreatic ribonuclease A.

To assess the role of the [65-72] disulfide bond in the oxidative folding of RNase A, use has been made of [C65S, C72S], a three-disulfide-containing mutant of RNase A which regenerates from its two-disulfide precursor in an oxidation and conformational folding-coupled rate-determining step. The distribution of disulfide bonds in the one-disulfide-containing ensemble of this mutant has been characterized. In general, the disulfide-bond distribution in its 1S ensemble agrees relatively well with the corresponding distribution in wt-RNase A and with distributions based on calculations of loop entropy, except for the absence of the [65-72] disulfide bond. There is no bias (over the entropic influence) for the three native disulfide bonds, [26-84], [40-95], and [58-110]. Previous oxidative folding results for wt-RNase A indicated the predominance of the des [40-95] intermediate over des [65-72] after the rate-determining step in the regeneration process. Considering that there is no preferential distribution of disulfides in the 1S ensemble of [C65S, C72S], in contrast to the preferential population of the [65-72] disulfide bond in wt-RNase A, these results indicate a critical role for the [65-72] disulfide bond in the regeneration of wt-RNase A. Furthermore, analysis of the disulfide distribution of the 1S intermediates of [C65S, C72S] compared to that of wt-RNase A lends support for a physicochemical basis for the previously observed slow folding rate of this mutant, compared to its analogue (des [65-72]) of wt-RNase A.

Amino Acid Substitution↗

PubMed2003

Shifting the competition between the intramolecular Reshuffling reaction and the direct oxidation reaction during the oxidative folding of kinetically trapped disulfide-insecure intermediates.

The oxidative folding pathway(s) of single-domain proteins can be characterized by the existence, stability, and structural nature of the intermediates that populate the regeneration pathway. Structured intermediates can be disulfide-secure in that they are able to protect their existing (native) disulfide bonds from SH/SS reshuffling and reduction reactions, and thereby form the native protein directly, i.e., by oxidation of their exposed (or locally exposable) thiols. Alternatively, they can be disulfide-insecure, usually requiring global unfolding to expose their free thiols. However, such an unfolding event also exposes the existing native disulfide bonds. Thus, the subsequent oxidation reaction to form the native protein in a disulfide-insecure intermediate competes with the intramolecular attack by the thiols of the macromolecule on its own native disulfide bonds, resulting in a large population of intermediates that are reshuffled instead of being oxidized. Under stabilizing conditions, disulfide-insecure species become long-lived kinetically trapped intermediates that slowly and only indirectly convert to the native protein through reshuffling reactions. In this study, trans-[Pt(en)(2)Cl(2)](2+), a strong oxidizing agent which has not traditionally been used in oxidative folding, was applied to shift the competition between reshuffling and oxidation reactions in des [58-110] and des [26-84], two long-lived disulfide-insecure intermediates of RNase A, and oxidize them directly under stable conditions to form the native protein. Such a successful direct conversion of kinetically trapped intermediates to the native molecule by trans-[Pt(en)(2)Cl(2)](2+) may be helpful in facilitating the oxidative folding processes of multi-disulfide-containing proteins in general.

Animals↗

PubMed2003

Characterizing the unstructured intermediates in oxidative folding.

A recently developed method is used here to characterize some of the folding intermediates, and the oxidative folding processes, of RNase A. This method is based on the ability of trans-[Pt(en)(2)Cl(2)](2+) to oxidize cysteine residues to form disulfide bonds faster than the disulfide bonds can be rearranged by reshuffling or reduction. Variations of this method have enabled us to address three issues. (i) How the nature of the residual structure and/or conformational order that is present, or develops, during the initial stages of folding can be elucidated. It is shown here that there is a 10-fold increase in the propensity of the unfolded reduced forms of RNase A to form the native set of disulfides directly, compared to the propensity under strongly denaturing conditions (4-6 M GdnHCl). Thus, the unfolded reduced forms of RNase A are not statistical coils with a more condensed form than in the GdnHCl-denatured state; rather, it is suggested that reduced RNase A has a little bias toward a native topology. (ii) The structural characterization of oxidative folding intermediates in terms of disulfide pairing is demonstrated; specifically, a lower-limit estimate is made of the percentage of native disulfide-containing molecules in the two-disulfide ensemble of RNase A. (iii) The critical role of structured intermediate species in determining the oxidative folding pathways of proteins was shown previously. Here, we demonstrate that the presence of a structured intermediate in the oxidative folding of proteins can be revealed by this method.

Chromatography, High Pressure Liquid↗

PubMed2003

Native conformational tendencies in unfolded polypeptides: development of a novel method to assess native conformational tendencies in the reduced forms of multiple disulfide-bonded proteins.

Oxidative folding is the concomitant formation of the native disulfide bonds and the native tertiary structure from the reduced and unfolded polypeptide. Of interest is the inherent conformational tendency (bias) present in the reduced polypeptide to dictate the formation of the full set of native disulfide bonds. Here, by application of a novel tool, we have been able to assess this "native conformational tendency" present in reduced and unfolded bovine pancreatic ribonuclease A (RNase A). The essence of this method lies in the ability of the oxidant [Pt(en)(2)Cl(2)](2+) (where "en" is ethylenediamine) to oxidize disulfide bonds under conditions in which both reduction and disulfide reshuffling, which are essential for rearranging non-native disulfide bonds, are extremely slow. When applied to RNase A, the method revealed little or no bias toward formation of the full native set of disulfide bonds in the fully reduced protein.

Animals↗

PubMed2002

Development of a novel method to populate native disulfide-bonded intermediates for structural characterization of proteins: implications for the mechanism of oxidative folding of RNase A.

RNase A, a model protein for oxidative folding studies, has four native disulfide bonds. The roles of des [40-95] and des [65-72], the two native-like structured three-disulfide-bonded intermediates populated between 8 and 25 degrees C during the oxidative folding of RNase A, are well characterized. Recent work focuses on both the formation of these structured disulfide intermediates from their unstructured precursors and on the subsequent oxidation of the structured species to form the native protein. The major obstacles in this work are the very low concentration of the precursor species and the difficulty of isolating some of the structured intermediates. Here, we demonstrate a novel method that enables the native disulfide-bonded intermediates to be populated and studied regardless of whether they have stable structure and/or are present at low concentrations during the oxidative folding or reductive unfolding process. The application of this method enabled us to populate and, in turn, study the key intermediates with two native disulfide bonds on the oxidative folding pathway of RNase A; it also facilitated the isolation of des [58-110] and des [26-84], the other two native-like structured des species whose isolation had thus far not been possible.

Animals↗