Modification Of Carboxyl Groups Research Articles

A Comparable Study on the Effects of Pristine and Functionalized Single-Walled Carbon Nanotubes on Schwann Cells

A comparable study on the effects of pristine and functionalized single-walled carbon nanotubes on schwann cells were conducted. Schwann cells were co-cultured with three types of SWNTs, purified raw SWNTs (C), hydroxyl purified SWNTs (C-OH) and carboxyl purified SWNTs (C-COOH) at 25μg/mL. C-COOH promoted the cell viability after 72h exposure and sustained its redox states by enhancing GPx expression. On the contrary, C and C-OH weaken the antioxidant activity due to the inhibition in main antioxidant enzymes and further led to the decline in cell viability. Carboxyl modification of the SWNTs helps to improve the biocompatibility of the SWNTs by strengthening the antioxidant ability.

Carboxylic group modification as a method of intensification of diuretic properties of 7-hydroxy-5-oxo-2,3-dihydro-1h,5h-pyrido[3,2,1-ij]quinoline-6-carboxylic acid 2-carboxyanilide

The intensive and purposeful search of fundamentally new diuretics conducted by our research laboratory among the derivatives of 4-hydroxyquinolin-2-ones has found 7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyrido[3,2,1-ij] quinoline-6-carboxylic acid 2-carboxyanilide as an intermediate lead compound. The basis for it is a high diuretic activity and a low toxicity of this substance. However, a free carboxylic group, which is often conductive to appearance of an extremely undesirable ulcerogenic action in relation to the gastro-intestinal tract, can be a serious obstacle for using the given compound as a drug. That is why we attempted to block the carboxylic group of 2-carboxyanilide by one of the known methods. For this purpose the methodology of both classical and non-classical bioisosteric replacements has been used. When conducting the synthetic part of the research the alternative ways of close structural analogues of the basic molecule were considered. As a result the optimal method for their obtaining has been offered; it differs by the minimal time costs and preparative high yields and purity of the target products. The structure of all substituted 7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyrido[3,2,1-ij] quinoline-6-carboxanilides synthesized has been confirmed by the data of elemental analysis, counter synthesis and NMR 1Н spectra. The primary pharmacological screening in studying the effect of the compounds obtained on the excretory function of the kidneys has been carried out in white outbread rats using the standard method. The testing has been performed in parallel and in comparison with Hydrochlorothiazide, as well as the abovementioned 2-carboxyanilide. It has been determined that some compounds under study in the dose of 10 mg/kg when introduced orally exceed not only Hydrochlorothiazide, but the basic molecule as well. By the results of the experiments carried out the structural and biological regularities that are important for further research have been found. As a new lead compound 7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinoline-6-carboxylic acid 2-carbamoylanilide has been suggested. It has no free COOH-group and, therefore, is not capable of having an great effect on the gastro-intestinal tract.

Influence of ssDNA Immobilization on the Conductance of Solution Gated Graphene Transistors

Carboxyl-modified graphene materials in both oxide and reduced state were explored in parallel for the preparation of field-effect transistors (FET). They were solution gated by phosphate buffer solution (PBS) (pH 7.2). Their conductance were examined and compared with unmodified graphene transistors, firstly. Then, after single strand DNA molecules were immobilized on reduced and oxide graphene transistors, their conductance and compared. Here ssDNA molecules were amino-tagged at the terminal five. It was found that ambipolar characteristic was exhibited by reduced graphene transistors, even they were undergone carboxyl modification. And it was also discovered that there were opposite conductance variation with the increasing of ssDNA concentrations and bigger changes were obtained by reduced carboxyl-modified graphene transistors.

Histone induced platelet aggregation is inhibited by normal albumin

IntroductionHistones are small, nuclear proteins that serve to package DNA. Recent reports suggest that extracellular histones, including histone H4, may contribute to the pathogenesis of sepsis; they promote platelet aggregation and thrombosis when released into the circulation during inflammation or cell death. The mechanisms by which the body minimizes the deleterious effects of circulating histones are unclear. Because histones can bind to plasma proteins, including albumin, we hypothesized that normal albumin can prevent histones from activating platelets. Materials and MethodsPlatelets and platelet-free plasma were obtained from healthy, adult subjects. The dose-dependent effects of histone H4 on platelet aggregation were studied by optical aggregometry. The effects of native and albumin-depleted plasma (prepared by affinity chromatography) on histone-induced platelet aggregation were also assessed. The effects of normal and surface-neutralized albumin (through modification of carboxyl groups) on histone-induced platelet activation and aggregation were evaluated using flow cytometry and aggregometry. ResultsHistone H4 induced platelet aggregation in a dose-dependent manner. This histone-induced platelet aggregation was inhibited by both plasma and human serum albumin in a dose-dependent fashion. Furthermore, depletion of albumin from plasma reduced its ability to inhibit aggregation. Finally, surface neutralization of albumin decreased its ability to inhibit histone-induced activation and aggregation. DiscussionThese data suggest that normal albumin serves a role in preventing histone-induced platelet aggregation in a charge-dependent manner.

Abstract 353: Diabetes Induces Increased Expression of Matricellular Osteoblast Factor Periostin and Inhibits the Calcification Inhibitor Matrix Gla Protein (mgp) in Rat Aorta

It is increasingly recognized that diabetes is associated with increased risk of cardiovascular events and vascular calcification. To understand the mechanism of vascular calcification in diabetes we used a rat model of type 2 diabetes. Rats were fed ad libitum with diet containing 45 kcal % fat (soybean oil and lard with 0.95 mg cholesterol /gram of lard), 35 kcal % carbohydrates and 20% kcal protein (HF diet) for eight weeks, followed by a single low dose of stz (30mg/kg intra peritoneally). Rats were then allowed to developed diabetes for sixteen weeks. Average blood glucose in diabetic HF rats 517.6mg/dl vs 87.8mg/dl in HF rats. The expression and immunohistochemical changes were analyzed in aorta of these rats after sixteen weeks of diabetes. Conformational specific gamma carboxylated (Gla) and undercarboxylated (Glu) MGP antibodies were used to assess the expression of Gla and Glu MGP in control and diabetic HF rat aorta. Western blot analysis show significantly reduced expression of Gla MGP in diabetic HF rat aorta and higher Glu MGP expression compared to control rat aorta suggesting that diabetic conditions inhibit the gamma carboxylation of MGP in aorta, gamma carboxylation modification is necessary for the MGP to work as physiological calcification inhibitor. Similarly, immunohistochemical analysis of diabetic HF rat and control rat aorta show increased staining of Glu MGP than Gla MGP compared to control rat aorta further confirm our Western data that modification of Gla MGP is inhibited in diabetic HF rats. Furthermore, immuno-histochemical data also show significantly increased in diabetic HF rat aorta expression of Periostin a matricellular osteoblast specific factor expression compared to control rat aorta. Our findings suggest that active diabetes impair the physiological properties of calcification inhibitor Gla MGP in aorta and increases the osteogenic factors in diabetic HF rats. Increased expression of osteoblastic factor and inhibition of Gla MGP suggests that diabetic HF rat aorta is under active transformation to become more bone like, precursor of arterial calcification.

Carboxyl Modification of Sugarcane Bagasse Hemicelluloses With Succinic Anhydride in Aqueous Solution

Abstract In this paper, we describe the modification of sugarcane bagasse with succinic anhydride to introduce carboxylic functions to sugarcane bagasse. The properties of the sugarcane bagasse hemicelluloses change after carboxyl modification. The influences of the chemical reaction on the conditions, the chemical composition, and the thermal stability of the modified product are described. One gram of dried sugarcane bagasse was modified using 3.0 g succinic anhydride and 0.1 g N-bromination succinic imide with a reaction time of 6 h at 70°C. The obtained materials were characterized via Fourier transform infrared spectroscopy, nuclear magnetic resonance, thermal analysis, and scanning electron microscopy.

Fluorescent Dye-Doped Silica Nanoparticles with Polyclonal Antibodies for the Rapid Detection of Salmonella spp.

This study investigated a novel detection method of Salmonella spp. based on immunofluorescence microscopy using fluorescent dye-doped silica nanoparticles (FDS-NPs). Tetraethyl orthosilicate (TEOS) was used as a precursor. The FDS-NPs produced with microemulsion combined with sol-gel techniques were spherical in shape and 40-70 nm in diameter. The observation of the particles under fluorescence microscope showed high intensity orange color luminescent (Rubpy dye) with high photostability. Surface modifications for bioconjugation with polyclonal antibodies were performed by two methods, amine and carboxyl group modifications. The FDS-NPs were coated with polyclonal antibodies (purified IgGs) against Salmonella spp. Results of bacterial detection by FDSNPs indicated that nanoparticles with amine modified surface could attach onto the cells of the test organisms, Salmonella weltevreden and S. enteritidis, producing a distinctively bright color under fluorescence microscope. However, the nanoparticles with carboxyl group modification alone did not bind well with the test organisms. The FDS-NPs developed here show a high potential for use in the rapid detection of Salmonella and other bio-molecules.

Carboxylic acid functionalization prevents the translocation of multi-walled carbon nanotubes at predicted environmentally relevant concentrations into targeted organs of nematode Caenorhabditis elegans

Carboxyl (-COOH) surface modified multi-walled carbon nanotubes (MWCNTs-COOH) can be used for targeted delivery of drugs and imaging. However, whether MWCNTs-COOH at environmentally relevant concentrations exert certain toxic effects on multicellular organisms and the underlying mechanisms are still largely unclear. In the present study, we applied the nematode Caenorhabditis elegans to evaluate the properties of MWCNTs-COOH at environmentally relevant concentrations by comparing the effects of MWCNTs and MWCNTs-COOH exposure on C. elegans from L1-larvae to adult at concentrations of 0.001-1000 μg L(-1). Exposure to MWCNTs could potentially damage the intestine (primary targeted organ) at concentrations greater than 0.1 μg L(-1) and functions of neurons and reproductive organ (secondary targeted organs) at concentrations greater than 0.001 μg L(-1). Carboxyl modification prevented the toxicity of MWCNTs on the primary and the secondary targeted organs at concentrations less than 100 μg L(-1), suggesting that carboxyl modification can effectively prevent the adverse effects of MWCNTs at environmentally relevant concentrations. After exposure, MWCNTs-COOH (1 mg L(-1)) were translocated into the spermatheca and embryos in the body through the primary targeted organs. However, MWCNTs-COOH (10 μg L(-1)) were not observed in spermatheca and embryos in the body of nematodes. Moreover, relatively high concentrations of MWCNTs-COOH exposed nematodes might have a hyper-permeable intestinal barrier, whereas MWCNTs-COOH at environmentally relevant concentrations effectively sustained the normally permeable state for the intestinal barrier. Therefore, we elucidated the cellular basis of carboxyl modification to prevent toxicity of MWCNTs at environmentally relevant concentrations. Our data highlights the key role of biological barriers in the primary targeted organs to block toxicity formation from MWCNTs, which will be useful for the design of effective prevention strategies against MWCNTs toxicity.

Preparation and characterisation of blends of poly(ethylene oxide) and functionalised epoxidised natural rubber

Blending of polymers has been a popular technique for making new materials with improved properties or processability. The synergistic properties in the blends are related to the miscibility of the components when there are significant interactions between the constituents. This paper reports the preparation of reactive blends of poly(ethylene oxide) (PEO) and acetic acid-modified epoxidised natural rubber (ENR50, which contains 50% of the isoprene units converted to epoxide groups) by solution casting. The carboxylic acid modification of ENR50 was carried out by reacting the ENR50 dissolved in toluene with excess acetic acid at 100°C. Ring-opening of epoxide group by acetic acid has led to an increase in the Tg. The effects of blend ratio of the acetic acid-modified ENR50 and PEO on the thermal properties were studied. The initial ENR50 has a Tg of −29°C and the acetic acid-modified ENR50 has led to a new Tg of 10°C. FTIR results showed that there was no chemical reaction between the acetic acid-modified ENR50 and PEO in the blends. This is in agreement with the DSC results where two distinct Tgs were observed in the blends.

Nitrilases in nitrile biocatalysis: recent progress and forthcoming research

Over the past decades, nitrilases have drawn considerable attention because of their application in nitrile degradation as prominent biocatalysts. Nitrilases are derived from bacteria, filamentous fungi, yeasts, and plants. In-depth investigations on their natural sources function mechanisms, enzyme structure, screening pathways, and biocatalytic properties have been conducted. Moreover, the immobilization, purification, gene cloning and modifications of nitrilase have been dwelt upon. Some nitrilases are used commercially as biofactories for carboxylic acids production, waste treatment, and surface modification. This critical review summarizes the current status of nitrilase research, and discusses a number of challenges and significant attempts in its further development. Nitrilase is a significant and promising biocatalyst for catalytic applications.

Evidence of Carboxyl Modification of Hydrogen-Free Diamond-Like Carbon Films Assisted by Radio Frequency Plasma in Vacuum

Modification of hydrogen-free diamond-like carbon (DLC) is presented, with acrylic acid (AA) vapor carried into a vacuum chamber by argon and with the in situ assistance of low-power radio frequency (RF) plasma at a temperature below 100°C. Measured by atomic force microscopy (AFM) technique, the roughness (Ra) of the DLC was 1.063±0.040 nm. XPS and FT-IR spectra analysis showed that carboxyl groups were immobilized on the surface of the DLC films, with about 40% of carboxyl group area coverage. It was found that the RF plasma and reaction time are important in enhancing the modification rate and efficiency.

Chemical modification of lactase for immobilization on carboxylic acid-functionalized microspheres

Surface interactions between an enzyme and support influence the retention of activity after immobilization. Chemical modification of enzymes prior to immobilization may be used to alter these interactions and enhance activity retention. Lactase (A. oryzae) was covalently conjugated to P(S/V-COOH) microspheres, with surface carboxylic acid densities of 9 μeq/g and 137 μeq/g, using carbodiimide chemistry. Under optimum pH and temperature conditions, activity retention was greater when the enzyme was conjugated to microspheres containing a lower density of surface carboxylic acid groups (32% activity retention) than when the enzyme was conjugated to microspheres having a greater density of surface carboxylic acid groups (11% activity retention). Chemical modification of lactase carboxylic acid groups with glucosamine prior to immobilization was evaluated as a means to increase activity retention. Under optimal conditions, modification resulted in a 17% decrease in soluble enzyme activity compared to the native enzyme. However, immobilization of the modified enzyme yielded 85% and 64% activity retention after conjugation to microspheres with a lower and higher density of surface carboxylic acid groups, respectively. The results suggest that increases in surface carboxylic acid density on the carrier promote the loss of lactase activity after immobilization, and chemical modification of the enzyme with glucosamine provides a means to retain catalytic activity after attachment to these supports.

Pegylated Fluorescent Peptides as Substrates of Proteolytic Enzymes

In this work the efficient and simple method of improvement specificity and solubility of low molecular weight proteinase substrates is described. The series of fluorescent substrates of selected proteolytic enzymes (neutrophil elastase, cathepsin G and proteinase 3 along with human airway trypsin like protease) were synthesized and modified by selective pegylation by the attachment of 2-(2-(2-aminoethoxy)ethoxy)acetic acid. Modification of the C-terminal carboxyl group resulted in the decrease in the specificity constants (k(cat)/K(M)) for all obtained analogues. The covalent attachment of PEG to N-terminal amino group has the opposite effect, as the increase in specificity constant was observed for all studied compounds. This outcome was pronounced the most for proteinase 3 substrate PEG-ABZ-Tyr-Tyr-Abu-ANB-NH2, whose catalytic constant (k(cat)) increased over three fold. The introduction of PEG moieties at both C- and N-terminal yielded the substrates with lower specificity constants. For substrate (ABZ-Arg-Gln-Asp-Arg-ANB-NH2) the influence of the PEG chain length on its kinetic parameters was investigated. Elongation of the PEG chain at N-terminal of this peptide decreased the specificity constant. In addition to the effect of pegylation on the kinetic parameters of the studied substrates, the introduced modifications significantly improved their solubility in buffer solutions applied for enzymatic investigations.

Composite Microspheres for Separation of Plasmid DNA Decorated with MNPs through in Situ Growth or Interfacial Immobilization Followed by Silica Coating

Raspberry-like colloidal polymer/magnetite/silica composite microspheres were rationally fabricated based on in situ growth or interfacial immobilization of magnetic nanoparticles (MNPs) onto the polymer matrices and the followed sol-gel coating process. Monodisperse cross-linked poly(styrene-co-glycidyl methacrylate) microspheres were first prepared by surfactant-free emulsion polymerization, followed by surface modification of carboxyl or amine moieties through thiol-epoxy click chemistry. Then the carboxyl-modified microspheres were in situ decorated with MNPs through solvothermal process or chemical coprecipitation reaction. In parallel, incorporation of MNPs onto polymer matrices was also realized by the interaction of amine-modified polymer microspheres with carboxyl-capped MNPs based on the electrostatic interaction. The two pathways for synthesis of the composite microspheres decorated with MNPs were systematically investigated. Furthermore, the composite microspheres were coated with a thin layer of silica through a sol-gel process. The thus-produced magnetic composite microspheres with desirable magnetization (~23 emu/g) served as effective supports for high-payload plasmid DNA enrichment (~17 μg per mg of microspheres), much better than that of the commercial-available sample of SM1-015B (~12 μg per mg of SM1-015B), shedding lights on the potential advantages of the nanoplatforms for separation of bioactive entities.

Control of Enzyme–Solid Interactions via Chemical Modification

Electrostatic forces could contribute significantly toward enzyme-solid interactions, and controlling these charge-charge interactions while maintaining high affinity, benign adsorption of enzymes on solids is a challenge. Here, we demonstrate that chemical modification of the surface carboxyl groups of enzymes can be used to adjust the net charge of the enzyme and control binding affinities to solid surfaces. Negatively charged nanosolid, α-Zr(HPO(4))(2)·H(2)O (abbreviated as α-ZrP) and two negatively charged proteins, glucose oxidase (GO) and methemoglobin (Hb), have been chosen as model systems. A limited number of the aspartate and glutamate side chains of these proteins are covalently modified with tetraethylenepentamine (TEPA) to convert these negatively charged proteins into the corresponding positively charged ones (cationized). Cationized proteins retained their structure and activities to a significant extent, and the influence of cationization on binding affinities has been tested. Cationized GO, for example, showed 250-fold increase in affinity for the negatively charged α-ZrP, when compared to that of the unmodified GO, and cationized Hb, similarly, indicated 26-fold increase in affinity. Circular dichroism spectra showed that α-ZrP-bound cationized GO retained native-like structure to a significant extent, and activity studies showed that cationized GO/α-ZrP complex is ~2.5-fold more active than GO/α-ZrP. Cationized Hb/α-ZrP retained ~75% of activity of Hb/α-ZrP. Therefore, enzyme cationization enhanced affinities by 1-2 orders of magnitude, while retaining considerable activity for the bound biocatalyst. This benign, chemical control over enzyme charge provided a powerful new strategy to rationally modulate enzyme-solid interactions while retaining their biocatalytic properties.

Protein‐grafted carboxylic poly(ether sulfone) membranes: Preparation and characterization

AbstractCarboxylic poly(ether sulfone) membranes were prepared by a controlled acetylating and surface‐oxidating reaction followed by the grafting of bovine serum albumin (BSA) and bovine serum fibrinogen (BFG) onto the surfaces. Attenuated total reflection–Fourier transform infrared spectroscopy, X‐ray photoelectron spectroscopy, and Micro BCA Protein Assay Kits confirmed that the proteins were successfully grafted onto the surfaces of the membranes. The protein grafting degrees were measured at different time intervals and under different conditions. The modified membranes showed higher hydrophilicity, lower protein (BSA and BFG) adsorption, and suppressed platelet adhesion values. Because of the binding of calcium ions in blood, the modified membranes showed longer plasma recalcification times, activated partial thromboplastin times, prothrombin times, and whole blood clotting times. The results indicate that the blood compatibility of the poly (ether sulfone) membranes could be improved after surface carboxylic modification and protein immobilization and that the modified membranes could be used in the blood purification field. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

4-Hydroxybenzyl Modification of the Highly Teratogenic Retinoid, 4-[(1E)-2-(5,5,8,8-Tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propen-1-yl]benzoic Acid (TTNPB), Yields a Compound That Induces Apoptosis in Breast Cancer Cells and Shows Reduced Teratogenicity

Retinoids are a class of compounds with structural similarity to vitamin A. These compounds inhibit the proliferation of many cancer cell lines but have had limited medical application as they are often toxic at therapeutic levels. Efforts to synthesize retinoids with a greater therapeutic index have met with limited success. 4-[(1E)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propen-1-yl]benzoic acid (TTNPB) is one of the most biologically active all-trans-retinoic acid (atRA) analogues and is highly teratogenic. In this study, we show that modification of the TTNPB carboxyl group with an N-(4-hydroxyphenyl)amido (4HPTTNPB) or a 4-hydroxybenzyl (4HBTTNPB) group changes the activity of the compound in cell culture and in vivo. Unlike TTNPB, both compounds induce apoptosis in cancer cells and bind poorly to the retinoic acid receptors (RARs). Like the similarly modified all-trans-retinoic acid (atRA) analogues N-(4-hydroxyphenyl)retinamide (4-HPR/fenretinide) and 4-hydroxybenzylretinone (4-HBR), 4HBTTNPB is a potent activator of components of the ER stress pathway. The amide-linked analogue, 4HPTTNPB, is less toxic to developing embryos than the parent TTNPB, and most significantly, the 4-hydroxybenzyl-modified compound (4HBTTNPB) that cannot be hydrolyzed in vivo to the parent TTNPB compound is nearly devoid of teratogenic liability.

Solubilization of Multi Walled Carbon Nanotubes Under a Facile and Mild Condition

Multi-walled carbon nanotubes (MWCNTs) functionalized with amino groups was prepared via chemical modification of carboxyl groups introduced on the carbon nanotube surfaces. Oxidation of MWCNTs was performed with ozone in aqueous phase and amidation of generated carboxylic groups, was occurred with amines in the presence of HATU as a coupling agent. Obtained functionalized MWCNTs are soluble in many common organic solvents. The functionalized MWCNTs were characterized in detail using FTIR-ATR, Raman CHN and SEM methods.

Effect of pre‐treatments on the biosorption of Chromium (VI) ions by the dead biomass of Rhizopus arrhizus

AbstractBACKGROUND: This work fulfils the need to develop an eco‐friendly biosorbent, elucidating the mechanism of biosorption. Removal of Cr(VI) by Rhizopus arrhizus was investigated in batch mode. Enhancement in the performance of the biosorbent was attempted by pre‐treating the biomass with inorganic and organic acids, chelating agent, cross‐linker and an organic solvent followed by autoclaving. The surface characterization of the biomass was carried out by potentiometric titration, surface area analysis, infrared spectroscopy, chemical modification of the biomass and scanning electron microscopy.RESULTS: All the physico‐chemical treatments of the biosorbent improved Cr(VI) uptake compared with the native biomass (21.72 mg g−1). The highest biosorption capacity (31.52 mg g−1) was achieved after pre‐treating the biomass with 0.5 mol L−1 HNO3 followed by autoclaving. Surface characterization of the biomass using pHzpc, potentiometry and Fourier transform infrared (FTIR) analysis revealed the role of amino and carboxyl groups in Cr(VI) removal by electrostatic attraction. Chemical modification of amino and carboxyl groups significantly decreased Cr(VI) uptake capacity confirming their role in biosorption. SEM analysis showed adsorption of Cr(VI) on the biosorbent surface.CONCLUSION: Rhizopus arrhizus biomass proved to be an effective and low cost alternative biosorbent for removal of Cr(VI) from aqueous solutions. Copyright © 2011 Society of Chemical Industry

Mass spectrometry-based carboxyl footprinting of proteins: Method evaluation

Protein structure determines function in biology, and a variety of approaches have been employed to obtain structural information about proteins. Mass spectrometry-based protein footprinting is one fast-growing approach. One labeling-based footprinting approach is the use of a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine ethyl ester (GEE) to modify solvent-accessible carboxyl groups on glutamate (E) and aspartate (D). This paper describes method development of carboxyl-group modification in protein footprinting. The modification protocol was evaluated by using the protein calmodulin as a model. Because carboxyl-group modification is a slow reaction relative to protein folding and unfolding, there is an issue that modifications at certain sites may induce protein unfolding and lead to additional modification at sites that are not solvent-accessible in the wild-type protein. We investigated this possibility by using hydrogen deuterium amide exchange (H/DX). The study demonstrated that application of carboxyl group modification in probing conformational changes in calmodulin induced by Ca2+ binding provides useful information that is not compromised by modification-induced protein unfolding.