Amit Mandal - Academia.edu (original) (raw)
Papers by Amit Mandal
The infection caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in ... more The infection caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in a pandemic with huge death toll and economic consequences. The virus attaches itself to the human epithelial cells through noncovalent bonding of its spike protein with the angiotensin-converting enzyme-2 (ACE2) receptor on the host cell. We hypothesized that perturbing the functionally active conformation of spike protein through reduction of its solvent accessible disulfide bond, thereby disintegrating its structural architecture, may be a feasible strategy to prevent infection. Proteomics data showed that N-acetyl cysteine (NAC), an antioxidant and mucolytic agent been widely in use in clinical medicine, forms covalent conjugates with solvent accessible cysteine residues of spike protein that were disulfide bonded in the native state. In silico analysis indicated that this covalent conjugation perturbed the stereo specific orientations of the interacting key residues of spike protein ...
Benign prostatic hyperplasia (BPH) and prostate cancer are the most frequently diagnosed conditio... more Benign prostatic hyperplasia (BPH) and prostate cancer are the most frequently diagnosed conditions in men above 60 years. BPH manifests as benign enlargement of the prostate gland causing pain and difficulty in micturition, often associated with other lower urinary tract symptoms. On the other hand, prostate cancer might initially set in as a tumor of no clinical significance, but can ultimately develop into an aggressively metastatic cancer at later stages. Due to overlapping symptomatic manifestations with BPH, it might be difficult to diagnose prostate cancer and differentiate it from BPH. Screening for prostate cancer is based on examining the levels of prostate specific antigen (PSA), a clinical biomarker for prostate cancer in blood. However, several reported cases indicate that PSA might lack the sensitivity and specificity required to differentiate between the cancerous and benign conditions of prostate. Therefore, in the absence of non-invasive biomarkers in a diagnostic s...
The FEBS Journal, 2019
Glycated hemoglobin (GHb) is the indicator of the long‐term glycemic index of an individual. GHb ... more Glycated hemoglobin (GHb) is the indicator of the long‐term glycemic index of an individual. GHb is formed by the irreversible modification of N‐terminal α‐amino group of β globin chain with glucose via Amadori rearrangement. Cation exchange chromatography exploits the difference in surface charges between GHb and native hemoglobin (HbA0) for their separation and quantification. However, glucose condensation is specific to primary amino groups. Therefore, structural characterization of GHb synthesized in vivo is essential as multiple glycation may interfere with GHb assessment. The stoichiometric composition of different glycated hemoglobin from a 19% GHb sample was deduced using native mass spectrometry. We observed a comparable population of α and β glycated tetramers for mono‐glycated HbA0. Surprisingly, doubly and triply glycated HbA0 also showed mono‐glycated α and β globins. Thus, we propose that glycation of hemoglobin (HbA) occurs symmetrically across α and β globins with pr...
Molecular microbiology, Jan 18, 2018
The SoxXAYZB(CD) -mediated pathway of bacterial sulfur-chemolithotrophy explains the oxidation of... more The SoxXAYZB(CD) -mediated pathway of bacterial sulfur-chemolithotrophy explains the oxidation of thiosulfate, sulfide, sulfur and sulfite but not tetrathionate. Advenella kashmirensis, which oxidizes tetrathionate to sulfate, besides forming it as an intermediate during thiosulfate oxidation, possesses a soxCDYZAXOB operon. Knock-out mutations proved that only SoxBCD is involved in A. kashmirensis tetrathionate oxidation, whereas thiosulfate-to-tetrathionate conversion is Sox independent. Expression of two glutathione metabolism-related proteins increased under chemolithotrophic conditions, as compared to the chemoorganotrophic one. Substrate-dependent oxygen consumption pattern of whole cells, and sulfur-oxidizing enzyme activities of cell-free extracts, measured in the presence/absence of thiol inhibitors/glutathione, corroborated glutathione involvement in tetrathionate oxidation. Furthermore, proteome analyses detected a sulfite:acceptor oxidoreductase (SorAB) exclusively under...
Asia Pacific journal of clinical nutrition, 2018
Nutritional anemia is a significant public health issue with 50-80% prevalence in Indian children... more Nutritional anemia is a significant public health issue with 50-80% prevalence in Indian children. Fortification of food, specifically milk, with iron is a potential approach to increase dietary iron intake. Ferric pyrophosphate [Fe4(P2O7)3] is organoleptically neutral and is less soluble in acid medium and, further, has low bioavailability in milk. However, since ascorbic acid is a potent enhancer of iron absorption, the coadministration of ascorbic acid with Fe4(P2O7)3 might enhance the absorption of iron. We evaluated the effect of ascorbic acid on iron absorption from a Fe4(P2O7)3 and an ascorbic acid fortified milk beverage with respect to milk fortified with Fe4(P2O7)3 alone. A double-blind, two-way crossover, randomized study was conducted in 25 mildly anemic children. The test group received milk fortified with beverage powder containing 7 mg isotopically labeled iron (57Fe/58Fe) as Fe4(P2O7)3, equimolar proportions of ascorbic acid and 200 mg of calcium whereas control grou...
Langmuir, 2017
published as an ASAP article. Note that technical editing may introduce minor changes to the manu... more published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these "Just Accepted" manuscripts. Adsorption induced changes of human hemoglobin on ferric pyrophosphate nanoparticle surface probed by isotope exchange mass spectrometry: An implication on structure-function correlation
Toxicology Reports, 2016
Cigarette smoke (CS) is an important source of morbidity and early mortality worldwide. Besides c... more Cigarette smoke (CS) is an important source of morbidity and early mortality worldwide. Besides causing various life-threatening diseases, CS is also known to cause hypoxia. Chronic hypoxia would induce early aging and premature death. Continuation of smoking during pregnancy is a known risk for the unborn child. Although carbon monoxide (CO) is considered to be a cause of hypoxia, the effect of other component(s) of CS on hypoxia is not known. Here we show by immunoblots and mass spectra analyses that in smoker's blood p-benzoquinone (p-BQ) derived from CS forms covalent adducts with cysteine 93 residues in both the  chains of hemoglobin (Hb) producing Hb-p-BQ adducts. UV-vis spectra and CD spectra analyses show that upon complexation with p-BQ the structure of Hb is altered. Compared to nonsmoker's Hb, the content of ␣-helix decreased significantly in smoker's Hb (p = 0.0224). p-BQ also induces aggregation of smoker's Hb as demonstrated by SDS-PAGE, dynamic light scattering and atomic force microscopy. Alteration of Hb structure in smoker's blood is accompanied by reduced oxygen binding capacity. Our results provide the first proof that p-BQ is a cause of hypoxia in smokers. We also show that although both p-BQ and CO are responsible for causing hypoxia in smokers, exposure to CO further affects the function over and above that produced by Hb-p-BQ adduct.
![Research paper thumbnail of Characterization of a Double Heterozygote HbE/β+ Thalassemia IVS 1-1 [G>T] in a Juvenile Diabetic](https://attachments.academia-assets.com/109901524/thumbnails/1.jpg)
](Indian Journal of Clinical Biochemistry, 2014
The present case report describes the molecular and proteomic based study of Hb variant HbE assoc... more The present case report describes the molecular and proteomic based study of Hb variant HbE associated with b ? thalassemia IVS 1-1 G[T, in a juvenile diabetic patient. Given the ethnic origin and mobility of the variant hemoglobin at alkaline pH, HbE would be suspected. But hematologically and clinically abnormality being detected, HPLC and Electrophoresis not being able to characterize due to retention time and band being in region of HbA2, respectively, further characterization of hemoglobinopathy was made using MALDI and IVS 1-1 G[T being validated by reverse dot blot hybridization. Capillary electrophoresis was also employed in order to separate HbE and HbA2 bands. This case report being first of its kind, wherein a HbE/b ? thalassemia has been characterized using multiple techniques.
Indian Journal of Clinical Biochemistry, 2012
The present report describes the molecular study of HbD Iran (beta) 22 Glu ? Gln associated with ... more The present report describes the molecular study of HbD Iran (beta) 22 Glu ? Gln associated with b-Thalassemia IVS1-5 (G [ C) found in India, and the first case in which mutation has been identified using mass spectrometry. Given the apparent ethnic origin and the mobility of the variant hemoglobin at alkaline pH, hemoglobin D-Punjab would be suspected, but HPLC excluded this possibility. Further characterization of hemoglobinopathy was made by using nondenaturing gel electrophoresis and matrix assisted laser desorption ionization mass spectrometry and IVS1-5 being validated by reverse dot blot hybridization followed by sequencing of the b-globin gene.
Journal of Biological Chemistry, 2003
The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia... more The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia coli (GlnRS) in 0.25 M potassium L-glutamate, a naturally occurring osmolyte in E. coli, has been studied. Both the native to molten globule and molten globule to unfolded state transitions are shifted significantly toward higher urea concentrations in the presence of L-glutamate, suggesting that L-glutamate has the ability to counteract the denaturing effect of urea. D-Glutamate has a similar effect on the equilibrium denaturation of glutaminyl-tRNA synthetase, indicating that the effect of L-glutamate may not be due to substrate-like binding to the native state. The activation energy of unfolding is not significantly affected in the presence of 0.25 M potassium L-glutamate, indicating that the native state is not preferentially stabilized by the osmolyte. Dramatic increase of coefficient of urea concentration dependence (m) values of both the transitions in the presence of glutamate suggests destabilization and increased solvent exposure of the denatured states. Four other osmolytes, sorbitol, trimethylamine oxide, inositol, and triethylene glycol, show either a modest effect or no effect on native to molten globule transition of glutaminyl-tRNA synthetase. However, glycine betaine significantly shifts the transition to higher urea concentrations. The effect of these osmolytes on other proteins is mixed. For example, glycine betaine counteracts urea denaturation of tubulin but promotes denaturation of S228N-repressor and carbonic anhydrase. Osmolyte counteraction of urea denaturation depends on osmolyte-protein pair.
Clinical Biochemistry, 2008
Objectives: The clinical analysis of hemoglobin by ion exchange chromatography can result in ambi... more Objectives: The clinical analysis of hemoglobin by ion exchange chromatography can result in ambiguities in identification of the nature of the globin chain present in patient samples. LC/ESI-MS provides rapid and precise determination of globin chain masses. Design and methods: Hemolysate of hemoglobin Q-India and hemoglobin S/D/F have been analyzed using ESI-MS. Tandem-MS has been used to establish mutation in α chain of hemoglobin Q. Results: The identification of hemoglobin Q-India is readily achieved by LC/ESI-MS, which establishes the presence of a mutant α chain differing in mass from normal α chain by 22 Da. The site of mutation has been identified by tandem-MS analysis of a tryptic fragment encompassing residues αV62-K90. LC/ESI-MS screening has also provide an example of simultaneous occurrence of mutant globin chains containing β6E → V (Hb S, sickle) and β121E → Q (Hb D) variant. Expression of γ G globin chain is also demonstrated in this sample. Conclusions: The site of mutation in hemoglobin Q-India is identified as α64D → H which differs from mutations α74D → H in Hb Q-Thailand and α75D → H in Hb Q-Iran. Mass spectrometric analysis of hemoglobins from a patient and her parents suggests inheritance of mutant β globin genes from both parents.
Analytical Biochemistry, 2019
Glutaredoxin 1 up-regulates deglutathionylation of alpha4 integrin and thereby restricts neutroph... more Glutaredoxin 1 up-regulates deglutathionylation of alpha4 integrin and thereby restricts neutrophil mobilization from bone marrow alpha4 integrin Patra, K. K., et al. 2019 Molecular dynamics investigation of a redox switch in the anti-HIV protein SAMHD1 SAMHD1 Niazi, A. K., et al.
The infection caused by Severe Acute Respiratory Syndrome–CoronaVirus-2 (SARS-CoV-2) resulted in ... more The infection caused by Severe Acute Respiratory Syndrome–CoronaVirus-2 (SARS-CoV-2) resulted in a pandemic across the globe with a huge death toll. The symptoms from SARS-CoV2 appear somewhat similar to the SARS-CoV-1 infection that appeared in early 21st century but the infectivity is far higher for the SARS-CoV-2. The virus attaches itself to exposed human epithelial cells through the spike protein. Recently discovered crystal structure of the complex of spike protein of SARS-CoV-2 with human angiotensin-converting enzyme 2 (ACE2) receptor indicated that the virus binds with the host cell very strongly. We hypothesized that the perturbation of the functionally active conformation of spike protein through the reduction of a solvent accessible disulfide bond (Cys391-Cys525) that provides its structural architecture, may 2 be a feasible strategy to disintegrate the spike protein from ACE2 receptor and thereby prevent the infection. Using in silico platform we showed that N-acetyl cy...
Biophysical chemistry, 2018
The self-assembly of intrinsically disordered protein tau into paired helical filament forms one ... more The self-assembly of intrinsically disordered protein tau into paired helical filament forms one of the hallmarks of Alzheimer's disease. However, the facets of innately disordered structure of tau and its conversion to a β-sheet-rich fibril during several tauopathies are poorly understood. Here, we provide a direct insight into the ensemble of highly heterogeneous conformational families of tau at physiological pH, by nano-electrospray mass spectrometry coupled with ion mobility. The average collision cross section of the most unfolded conformer was higher by >2 fold than that of the most folded one. Acidic pH largely induced unfolding in tau, obliterating the compact conformers completely. The highly unfolded conformers were the key species bestowing the unusual solubility to tau at low pH, with limited contribution from intramolecular long-range interfaces giving rise to ordered conformers. Contrarily, alkaline pH shifted tau towards folded conformations due to charge neut...
Chembiochem : a European journal of chemical biology, Jan 8, 2018
Electrostatic attraction between α and β globin chains hold the subunits together in tetrameric h... more Electrostatic attraction between α and β globin chains hold the subunits together in tetrameric human hemoglobin molecule (α₂β₂). Compared to normal globin chains, the affinity of a mutant chain to its partner globin in genetic variants of hemoglobin might be different. This leads to an unequal abundance of normal and variant hemoglobin in heterozygous sample, even though the rates of synthesis of both normal and variant chains are same. The aforementioned affinities across various globin chains might be assessed by the quantification of different forms of tetramers present in a variant hemoglobin sample. In the present study, exploiting mass differences between globin chains, differentially populated hemoglobin tetramers present in Hb Beckman, a β variant (βA135D), were structurally characterized. The relative populations of dissymmetric tetramers (α₂β₂, α₂ββV and α₂βV₂) indicated that both β and βV have differential affinities towards α globin chain. The conformational dynamics an...
Hemoglobin, Jan 6, 2017
In general, the reactivity of cysteine residues of proteins is measured by 5,5'-dithiobis(2-n... more In general, the reactivity of cysteine residues of proteins is measured by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) kinetics using spectrophotometry. Proteins with several cysteine residues may exhibit varying DTNB kinetics but residue level information can only be obtained with the prior knowledge of their three-dimensional structure. However, this method is limited in its application to the proteins containing chromophores having overlapping absorption profile with 2-nitro-5-thiobenzoic acid, such as hemoglobin (Hb). Additionally, this method is incapable of assigning cysteine reactivity at the residue levels of proteins with unknown crystal structures. However, a mass spectrometry (MS)-based platform might provide a solution to these problems. In the present study, alkylation kinetics of cysteine residues of adult human Hb (Hb A; α2β2) and sickle cell Hb (Hb S; HBB: c.20A>T) were investigated using matrix-assisted laser desorption/ionization (MALDI) MS. Differential site...
Analytical chemistry, Jan 11, 2015
To gain insight into the underlying mechanisms of various biological events, it is important to s... more To gain insight into the underlying mechanisms of various biological events, it is important to study the structure-function correlation of proteins within cells. Structural probes used in spectroscopic tools to investigate protein conformation are similar across all proteins. Therefore, structural studies are restricted to purified proteins in vitro and these findings are extrapolated in cells to correlate their functions in vivo. However, due to cellular complexity, in vivo and in vitro environments are radically different. Here, we show a novel way to monitor the structural transition of human hemoglobin upon oxygen binding in living red blood cells (RBCs), using hydrogen/deuterium exchange-based mass spectrometry (H/DX-MS). Exploiting permeability of D2O across cell membrane, the isotope exchange of polypeptide backbone amide hydrogens of hemoglobin was carried out inside RBCs and monitored using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). To explor...
Introduction and Objective: Suicide is a major cause of mortality and a significant health proble... more Introduction and Objective: Suicide is a major cause of mortality and a significant health problem globally. Discovery of a biological marker could help in diagnosing subjects at high risk of attempting suicide and understanding the molecular event associated with suicidal behaviour that might help in early intervention. Methods: In the present study differential expression of plasma proteins in suicidal attempters were analysed using two dimensional gel electrophoresis and protein identification was done using MALDI mass spectrometry. Results and Discussion: The differential expression analysis based on spot intensities in 2D gel showed Apolipoprotein-AIV, Retinol Binding Protein-4 and Complement C 4A protein in plasma were significantly down regulated in suicidal attempters compared to age and gender matched healthy controls. The clinical characterisation of suicidal attempters showed that all attempts were impulsive and in response to life stressors. Apo A-IV has multiple functio...
Hemoglobin, the most abundant protein in red blood cell, is the carrier of oxygen from lungs to t... more Hemoglobin, the most abundant protein in red blood cell, is the carrier of oxygen from lungs to tissues. Hemoglobin is a tetrameric protein, consisting four globin chains where each of the four globin chains is non-covalently linked to a prosthetic oxygen-binding heme group and Fe+2 ion tetradentated centrally by porphyrin ring. The various globin chain synthesis is controlled by different genes expression at different stages of life. In a normal healthy adult, approximately 95% of total hemoglobin is HbA0 (α2β2) with 2-3% being HbA2 (α2δ2) and about 1% is fetal hemoglobin HbF (α22). Mutation in globin gene may lead to substitution of an amino acid in globin polypeptide chain which results hemoglobin variant. In addition to point mutation, hemoglobin variants may exist with deletion/fusion in globin chains. Many of these hemoglobin variants lead to healthcare problem such as sickle cell disease, erythrocytosis, cyanosis etc. Thalassemia is an autosomal recessive disorder, involves ...
![Research paper thumbnail of Characterization of a hemoglobin variant: HbQ-India / IVS 1-1 [G>T]-β-thalassemia](https://attachments.academia-assets.com/78205751/thumbnails/1.jpg)
](Indian journal of clinical biochemistry : IJCB, 2010
Hemoglobin Q- India (alpha) 64 Asp → His is an alpha chain variant which is generally found in he... more Hemoglobin Q- India (alpha) 64 Asp → His is an alpha chain variant which is generally found in heterozygous state and presents normal hematological blood picture. Here we report a rare case of HbQ-India with a thalassemic phenotype that has been analyzed using a combination of mass spectrometry, gene sequencing and PCR analysis. This combined analyses revealed the HbQ variant to be associated with a beta chain mutation, IVS 1-1 [G>T]. Though HbQ has earlier been reported with thalassemic trait using different techniques, this is the first report of a compound α and β chain Hb heterozygous mutant involving HbQ and IVS1-1 being validated using Mass Spectrometry and Reverse dot blot hybridization.
The infection caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in ... more The infection caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in a pandemic with huge death toll and economic consequences. The virus attaches itself to the human epithelial cells through noncovalent bonding of its spike protein with the angiotensin-converting enzyme-2 (ACE2) receptor on the host cell. We hypothesized that perturbing the functionally active conformation of spike protein through reduction of its solvent accessible disulfide bond, thereby disintegrating its structural architecture, may be a feasible strategy to prevent infection. Proteomics data showed that N-acetyl cysteine (NAC), an antioxidant and mucolytic agent been widely in use in clinical medicine, forms covalent conjugates with solvent accessible cysteine residues of spike protein that were disulfide bonded in the native state. In silico analysis indicated that this covalent conjugation perturbed the stereo specific orientations of the interacting key residues of spike protein ...
Benign prostatic hyperplasia (BPH) and prostate cancer are the most frequently diagnosed conditio... more Benign prostatic hyperplasia (BPH) and prostate cancer are the most frequently diagnosed conditions in men above 60 years. BPH manifests as benign enlargement of the prostate gland causing pain and difficulty in micturition, often associated with other lower urinary tract symptoms. On the other hand, prostate cancer might initially set in as a tumor of no clinical significance, but can ultimately develop into an aggressively metastatic cancer at later stages. Due to overlapping symptomatic manifestations with BPH, it might be difficult to diagnose prostate cancer and differentiate it from BPH. Screening for prostate cancer is based on examining the levels of prostate specific antigen (PSA), a clinical biomarker for prostate cancer in blood. However, several reported cases indicate that PSA might lack the sensitivity and specificity required to differentiate between the cancerous and benign conditions of prostate. Therefore, in the absence of non-invasive biomarkers in a diagnostic s...
The FEBS Journal, 2019
Glycated hemoglobin (GHb) is the indicator of the long‐term glycemic index of an individual. GHb ... more Glycated hemoglobin (GHb) is the indicator of the long‐term glycemic index of an individual. GHb is formed by the irreversible modification of N‐terminal α‐amino group of β globin chain with glucose via Amadori rearrangement. Cation exchange chromatography exploits the difference in surface charges between GHb and native hemoglobin (HbA0) for their separation and quantification. However, glucose condensation is specific to primary amino groups. Therefore, structural characterization of GHb synthesized in vivo is essential as multiple glycation may interfere with GHb assessment. The stoichiometric composition of different glycated hemoglobin from a 19% GHb sample was deduced using native mass spectrometry. We observed a comparable population of α and β glycated tetramers for mono‐glycated HbA0. Surprisingly, doubly and triply glycated HbA0 also showed mono‐glycated α and β globins. Thus, we propose that glycation of hemoglobin (HbA) occurs symmetrically across α and β globins with pr...
Molecular microbiology, Jan 18, 2018
The SoxXAYZB(CD) -mediated pathway of bacterial sulfur-chemolithotrophy explains the oxidation of... more The SoxXAYZB(CD) -mediated pathway of bacterial sulfur-chemolithotrophy explains the oxidation of thiosulfate, sulfide, sulfur and sulfite but not tetrathionate. Advenella kashmirensis, which oxidizes tetrathionate to sulfate, besides forming it as an intermediate during thiosulfate oxidation, possesses a soxCDYZAXOB operon. Knock-out mutations proved that only SoxBCD is involved in A. kashmirensis tetrathionate oxidation, whereas thiosulfate-to-tetrathionate conversion is Sox independent. Expression of two glutathione metabolism-related proteins increased under chemolithotrophic conditions, as compared to the chemoorganotrophic one. Substrate-dependent oxygen consumption pattern of whole cells, and sulfur-oxidizing enzyme activities of cell-free extracts, measured in the presence/absence of thiol inhibitors/glutathione, corroborated glutathione involvement in tetrathionate oxidation. Furthermore, proteome analyses detected a sulfite:acceptor oxidoreductase (SorAB) exclusively under...
Asia Pacific journal of clinical nutrition, 2018
Nutritional anemia is a significant public health issue with 50-80% prevalence in Indian children... more Nutritional anemia is a significant public health issue with 50-80% prevalence in Indian children. Fortification of food, specifically milk, with iron is a potential approach to increase dietary iron intake. Ferric pyrophosphate [Fe4(P2O7)3] is organoleptically neutral and is less soluble in acid medium and, further, has low bioavailability in milk. However, since ascorbic acid is a potent enhancer of iron absorption, the coadministration of ascorbic acid with Fe4(P2O7)3 might enhance the absorption of iron. We evaluated the effect of ascorbic acid on iron absorption from a Fe4(P2O7)3 and an ascorbic acid fortified milk beverage with respect to milk fortified with Fe4(P2O7)3 alone. A double-blind, two-way crossover, randomized study was conducted in 25 mildly anemic children. The test group received milk fortified with beverage powder containing 7 mg isotopically labeled iron (57Fe/58Fe) as Fe4(P2O7)3, equimolar proportions of ascorbic acid and 200 mg of calcium whereas control grou...
Langmuir, 2017
published as an ASAP article. Note that technical editing may introduce minor changes to the manu... more published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these "Just Accepted" manuscripts. Adsorption induced changes of human hemoglobin on ferric pyrophosphate nanoparticle surface probed by isotope exchange mass spectrometry: An implication on structure-function correlation
Toxicology Reports, 2016
Cigarette smoke (CS) is an important source of morbidity and early mortality worldwide. Besides c... more Cigarette smoke (CS) is an important source of morbidity and early mortality worldwide. Besides causing various life-threatening diseases, CS is also known to cause hypoxia. Chronic hypoxia would induce early aging and premature death. Continuation of smoking during pregnancy is a known risk for the unborn child. Although carbon monoxide (CO) is considered to be a cause of hypoxia, the effect of other component(s) of CS on hypoxia is not known. Here we show by immunoblots and mass spectra analyses that in smoker's blood p-benzoquinone (p-BQ) derived from CS forms covalent adducts with cysteine 93 residues in both the  chains of hemoglobin (Hb) producing Hb-p-BQ adducts. UV-vis spectra and CD spectra analyses show that upon complexation with p-BQ the structure of Hb is altered. Compared to nonsmoker's Hb, the content of ␣-helix decreased significantly in smoker's Hb (p = 0.0224). p-BQ also induces aggregation of smoker's Hb as demonstrated by SDS-PAGE, dynamic light scattering and atomic force microscopy. Alteration of Hb structure in smoker's blood is accompanied by reduced oxygen binding capacity. Our results provide the first proof that p-BQ is a cause of hypoxia in smokers. We also show that although both p-BQ and CO are responsible for causing hypoxia in smokers, exposure to CO further affects the function over and above that produced by Hb-p-BQ adduct.
Indian Journal of Clinical Biochemistry, 2014
The present case report describes the molecular and proteomic based study of Hb variant HbE assoc... more The present case report describes the molecular and proteomic based study of Hb variant HbE associated with b ? thalassemia IVS 1-1 G[T, in a juvenile diabetic patient. Given the ethnic origin and mobility of the variant hemoglobin at alkaline pH, HbE would be suspected. But hematologically and clinically abnormality being detected, HPLC and Electrophoresis not being able to characterize due to retention time and band being in region of HbA2, respectively, further characterization of hemoglobinopathy was made using MALDI and IVS 1-1 G[T being validated by reverse dot blot hybridization. Capillary electrophoresis was also employed in order to separate HbE and HbA2 bands. This case report being first of its kind, wherein a HbE/b ? thalassemia has been characterized using multiple techniques.
Indian Journal of Clinical Biochemistry, 2012
The present report describes the molecular study of HbD Iran (beta) 22 Glu ? Gln associated with ... more The present report describes the molecular study of HbD Iran (beta) 22 Glu ? Gln associated with b-Thalassemia IVS1-5 (G [ C) found in India, and the first case in which mutation has been identified using mass spectrometry. Given the apparent ethnic origin and the mobility of the variant hemoglobin at alkaline pH, hemoglobin D-Punjab would be suspected, but HPLC excluded this possibility. Further characterization of hemoglobinopathy was made by using nondenaturing gel electrophoresis and matrix assisted laser desorption ionization mass spectrometry and IVS1-5 being validated by reverse dot blot hybridization followed by sequencing of the b-globin gene.
Journal of Biological Chemistry, 2003
The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia... more The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia coli (GlnRS) in 0.25 M potassium L-glutamate, a naturally occurring osmolyte in E. coli, has been studied. Both the native to molten globule and molten globule to unfolded state transitions are shifted significantly toward higher urea concentrations in the presence of L-glutamate, suggesting that L-glutamate has the ability to counteract the denaturing effect of urea. D-Glutamate has a similar effect on the equilibrium denaturation of glutaminyl-tRNA synthetase, indicating that the effect of L-glutamate may not be due to substrate-like binding to the native state. The activation energy of unfolding is not significantly affected in the presence of 0.25 M potassium L-glutamate, indicating that the native state is not preferentially stabilized by the osmolyte. Dramatic increase of coefficient of urea concentration dependence (m) values of both the transitions in the presence of glutamate suggests destabilization and increased solvent exposure of the denatured states. Four other osmolytes, sorbitol, trimethylamine oxide, inositol, and triethylene glycol, show either a modest effect or no effect on native to molten globule transition of glutaminyl-tRNA synthetase. However, glycine betaine significantly shifts the transition to higher urea concentrations. The effect of these osmolytes on other proteins is mixed. For example, glycine betaine counteracts urea denaturation of tubulin but promotes denaturation of S228N-repressor and carbonic anhydrase. Osmolyte counteraction of urea denaturation depends on osmolyte-protein pair.
Clinical Biochemistry, 2008
Objectives: The clinical analysis of hemoglobin by ion exchange chromatography can result in ambi... more Objectives: The clinical analysis of hemoglobin by ion exchange chromatography can result in ambiguities in identification of the nature of the globin chain present in patient samples. LC/ESI-MS provides rapid and precise determination of globin chain masses. Design and methods: Hemolysate of hemoglobin Q-India and hemoglobin S/D/F have been analyzed using ESI-MS. Tandem-MS has been used to establish mutation in α chain of hemoglobin Q. Results: The identification of hemoglobin Q-India is readily achieved by LC/ESI-MS, which establishes the presence of a mutant α chain differing in mass from normal α chain by 22 Da. The site of mutation has been identified by tandem-MS analysis of a tryptic fragment encompassing residues αV62-K90. LC/ESI-MS screening has also provide an example of simultaneous occurrence of mutant globin chains containing β6E → V (Hb S, sickle) and β121E → Q (Hb D) variant. Expression of γ G globin chain is also demonstrated in this sample. Conclusions: The site of mutation in hemoglobin Q-India is identified as α64D → H which differs from mutations α74D → H in Hb Q-Thailand and α75D → H in Hb Q-Iran. Mass spectrometric analysis of hemoglobins from a patient and her parents suggests inheritance of mutant β globin genes from both parents.
Analytical Biochemistry, 2019
Glutaredoxin 1 up-regulates deglutathionylation of alpha4 integrin and thereby restricts neutroph... more Glutaredoxin 1 up-regulates deglutathionylation of alpha4 integrin and thereby restricts neutrophil mobilization from bone marrow alpha4 integrin Patra, K. K., et al. 2019 Molecular dynamics investigation of a redox switch in the anti-HIV protein SAMHD1 SAMHD1 Niazi, A. K., et al.
The infection caused by Severe Acute Respiratory Syndrome–CoronaVirus-2 (SARS-CoV-2) resulted in ... more The infection caused by Severe Acute Respiratory Syndrome–CoronaVirus-2 (SARS-CoV-2) resulted in a pandemic across the globe with a huge death toll. The symptoms from SARS-CoV2 appear somewhat similar to the SARS-CoV-1 infection that appeared in early 21st century but the infectivity is far higher for the SARS-CoV-2. The virus attaches itself to exposed human epithelial cells through the spike protein. Recently discovered crystal structure of the complex of spike protein of SARS-CoV-2 with human angiotensin-converting enzyme 2 (ACE2) receptor indicated that the virus binds with the host cell very strongly. We hypothesized that the perturbation of the functionally active conformation of spike protein through the reduction of a solvent accessible disulfide bond (Cys391-Cys525) that provides its structural architecture, may 2 be a feasible strategy to disintegrate the spike protein from ACE2 receptor and thereby prevent the infection. Using in silico platform we showed that N-acetyl cy...
Biophysical chemistry, 2018
The self-assembly of intrinsically disordered protein tau into paired helical filament forms one ... more The self-assembly of intrinsically disordered protein tau into paired helical filament forms one of the hallmarks of Alzheimer's disease. However, the facets of innately disordered structure of tau and its conversion to a β-sheet-rich fibril during several tauopathies are poorly understood. Here, we provide a direct insight into the ensemble of highly heterogeneous conformational families of tau at physiological pH, by nano-electrospray mass spectrometry coupled with ion mobility. The average collision cross section of the most unfolded conformer was higher by >2 fold than that of the most folded one. Acidic pH largely induced unfolding in tau, obliterating the compact conformers completely. The highly unfolded conformers were the key species bestowing the unusual solubility to tau at low pH, with limited contribution from intramolecular long-range interfaces giving rise to ordered conformers. Contrarily, alkaline pH shifted tau towards folded conformations due to charge neut...
Chembiochem : a European journal of chemical biology, Jan 8, 2018
Electrostatic attraction between α and β globin chains hold the subunits together in tetrameric h... more Electrostatic attraction between α and β globin chains hold the subunits together in tetrameric human hemoglobin molecule (α₂β₂). Compared to normal globin chains, the affinity of a mutant chain to its partner globin in genetic variants of hemoglobin might be different. This leads to an unequal abundance of normal and variant hemoglobin in heterozygous sample, even though the rates of synthesis of both normal and variant chains are same. The aforementioned affinities across various globin chains might be assessed by the quantification of different forms of tetramers present in a variant hemoglobin sample. In the present study, exploiting mass differences between globin chains, differentially populated hemoglobin tetramers present in Hb Beckman, a β variant (βA135D), were structurally characterized. The relative populations of dissymmetric tetramers (α₂β₂, α₂ββV and α₂βV₂) indicated that both β and βV have differential affinities towards α globin chain. The conformational dynamics an...
Hemoglobin, Jan 6, 2017
In general, the reactivity of cysteine residues of proteins is measured by 5,5'-dithiobis(2-n... more In general, the reactivity of cysteine residues of proteins is measured by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) kinetics using spectrophotometry. Proteins with several cysteine residues may exhibit varying DTNB kinetics but residue level information can only be obtained with the prior knowledge of their three-dimensional structure. However, this method is limited in its application to the proteins containing chromophores having overlapping absorption profile with 2-nitro-5-thiobenzoic acid, such as hemoglobin (Hb). Additionally, this method is incapable of assigning cysteine reactivity at the residue levels of proteins with unknown crystal structures. However, a mass spectrometry (MS)-based platform might provide a solution to these problems. In the present study, alkylation kinetics of cysteine residues of adult human Hb (Hb A; α2β2) and sickle cell Hb (Hb S; HBB: c.20A>T) were investigated using matrix-assisted laser desorption/ionization (MALDI) MS. Differential site...
Analytical chemistry, Jan 11, 2015
To gain insight into the underlying mechanisms of various biological events, it is important to s... more To gain insight into the underlying mechanisms of various biological events, it is important to study the structure-function correlation of proteins within cells. Structural probes used in spectroscopic tools to investigate protein conformation are similar across all proteins. Therefore, structural studies are restricted to purified proteins in vitro and these findings are extrapolated in cells to correlate their functions in vivo. However, due to cellular complexity, in vivo and in vitro environments are radically different. Here, we show a novel way to monitor the structural transition of human hemoglobin upon oxygen binding in living red blood cells (RBCs), using hydrogen/deuterium exchange-based mass spectrometry (H/DX-MS). Exploiting permeability of D2O across cell membrane, the isotope exchange of polypeptide backbone amide hydrogens of hemoglobin was carried out inside RBCs and monitored using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). To explor...
Introduction and Objective: Suicide is a major cause of mortality and a significant health proble... more Introduction and Objective: Suicide is a major cause of mortality and a significant health problem globally. Discovery of a biological marker could help in diagnosing subjects at high risk of attempting suicide and understanding the molecular event associated with suicidal behaviour that might help in early intervention. Methods: In the present study differential expression of plasma proteins in suicidal attempters were analysed using two dimensional gel electrophoresis and protein identification was done using MALDI mass spectrometry. Results and Discussion: The differential expression analysis based on spot intensities in 2D gel showed Apolipoprotein-AIV, Retinol Binding Protein-4 and Complement C 4A protein in plasma were significantly down regulated in suicidal attempters compared to age and gender matched healthy controls. The clinical characterisation of suicidal attempters showed that all attempts were impulsive and in response to life stressors. Apo A-IV has multiple functio...
Hemoglobin, the most abundant protein in red blood cell, is the carrier of oxygen from lungs to t... more Hemoglobin, the most abundant protein in red blood cell, is the carrier of oxygen from lungs to tissues. Hemoglobin is a tetrameric protein, consisting four globin chains where each of the four globin chains is non-covalently linked to a prosthetic oxygen-binding heme group and Fe+2 ion tetradentated centrally by porphyrin ring. The various globin chain synthesis is controlled by different genes expression at different stages of life. In a normal healthy adult, approximately 95% of total hemoglobin is HbA0 (α2β2) with 2-3% being HbA2 (α2δ2) and about 1% is fetal hemoglobin HbF (α22). Mutation in globin gene may lead to substitution of an amino acid in globin polypeptide chain which results hemoglobin variant. In addition to point mutation, hemoglobin variants may exist with deletion/fusion in globin chains. Many of these hemoglobin variants lead to healthcare problem such as sickle cell disease, erythrocytosis, cyanosis etc. Thalassemia is an autosomal recessive disorder, involves ...
Indian journal of clinical biochemistry : IJCB, 2010
Hemoglobin Q- India (alpha) 64 Asp → His is an alpha chain variant which is generally found in he... more Hemoglobin Q- India (alpha) 64 Asp → His is an alpha chain variant which is generally found in heterozygous state and presents normal hematological blood picture. Here we report a rare case of HbQ-India with a thalassemic phenotype that has been analyzed using a combination of mass spectrometry, gene sequencing and PCR analysis. This combined analyses revealed the HbQ variant to be associated with a beta chain mutation, IVS 1-1 [G>T]. Though HbQ has earlier been reported with thalassemic trait using different techniques, this is the first report of a compound α and β chain Hb heterozygous mutant involving HbQ and IVS1-1 being validated using Mass Spectrometry and Reverse dot blot hybridization.