Deletion of the carboxyl‐terminal residue disrupts the amino‐terminal folding,self‐association,and thermal stability of an amphipathic antimicrobial peptide

Understanding the complex relationship between amino acid sequence and protein behaviors, such as folding and self‐association, is a major goal of protein research. In the present work, we examined the effects of deleting a C‐terminal residue on the intrinsic properties of an amphapathic α‐helix of mastoparan‐B (MP‐B), an antimicrobial peptide with the sequence LKLKSIVSWAKKVL‐NH2. We used circular dichroism and nuclear magnetic resonance to demonstrate that the peptide MP‐B^[1‐13] displayed significant unwinding at the N‐terminal helix compared with the parent peptide of MP‐B, as the temperature increased when the residue at position 14 was deleted. Pulsed‐field gradient nuclear magnetic resonance data revealed that MP‐B forms a larger diffusion unit than MP‐B^[1‐13] at all experimental temperatures and continuously dissociates as the temperature increases. In contrast, the size of the diffusion unit of MP‐B^[1‐13] is almost independent of temperature. These findings suggest that deleting the flexible, hydrophobic amino acid from the C‐terminus of MP‐B is sufficient to change the intrinsic helical thermal stability and self‐association. This effect is most likely because of the modulation of enthalpic interactions and conformational freedom that are specified by this residue. Our results implicate terminal residues in the biological function of an antimicrobial peptide. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Quaternary conformational stability: The effect of reversible self‐association on the fibrillation of two insulin analogs

Under conditions relevant to the manufacturing of insulin (e.g., pH 3, room temperature), biosynthetic human insulin (BHI), and Lispro insulin (Lispro) require a nucleation step to initiate aggregation. However, upon seeding with preformed aggregates, both insulins rapidly aggregate into nonnative fibrils. Far ultraviolet circular dichroism (far‐UV CD) and second derivative Fourier transform infrared (2D‐FTIR) spectroscopic analyses show that the fibrillation process involves a change in protein secondary structure from α‐helical in native insulin to predominantly β‐sheet in the nonnative fibrils. After seeding, Lispro aggregates faster than BHI, likely because of a reduced propensity to reversibly self‐associate. Composition gradient multi‐angle light scattering (CG‐MALS) analyses show that Lispro is more monomeric than BHI, whereas their conformational stabilities measured by denaturant‐induced unfolding are statistically indistinguishable. For both BHI and Lispro, as the protein concentration increases, the apparent first‐order rate constant for soluble protein loss decreases. To explain these phenomena, we propose an aggregation model that assumes fibril growth through monomer addition with competitive inhibition by insulin dimers. Biotechnol. Bioeng. 2011;108: 2359–2370. © 2011 Wiley Periodicals, Inc.     

Interaction of cationic dodecyl‐trimethyl‐ammonium bromide with oxy‐HbGp by isothermal titration and differential scanning calorimetric studies: Effect of proximity of isoelectric point

In this work, isothermal titration and differential scanning calorimetric methods, in combination with pyrene fluorescence emission and dynamic light scattering have been used to investigate the interaction of dodecyltrimethylammonium bromide (DTAB) with the giant extracellular Glossoscolex paulistus hemoglobin (HbGp) in the oxy‐form, at pH values around the isoelectric point (pI ≈ 5.5). Our ITC results have shown that the interaction of DTAB with the hemoglobin is more intense at pH 7.0, with a smaller cac (critical aggregation concentration) value. The increase of protein concentration does not influence the cac value of the interaction, at both pH values. Therefore, the beginning of the DTAB‐oxy‐HbGp premicellar aggregates formation, in the cac region, is not affected by the increase of protein concentration. HSDSC studies show higher T_m values at pH 5.0, in the absence and presence of DTAB, when compared with pH 7.0. Furthermore, at pH 7.0, an aggregation process is observed with DTAB in the range from 0.75 to 1.5 mmol/L, noticed by the exothermic peak, and similar to that observed for pure oxy‐HbGp, at pH 5.0, and in the presence of DTAB. DLS melting curves show a decrease on the hemoglobin thermal stability for the oxy‐HbGp‐DTAB mixtures and formation of larger aggregates, at pH 7.0. Our present data, together with previous results, support the observation that the protein structural changes, at pH 7.0, occur at smaller DTAB concentrations, as compared with pH 5.0, due to the acidic pI of protein that favors the oxy‐HbGp‐cationic surfactant interaction at neutral pH. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 199–211, 2016.     

Effects of water activity and aqueous solvent ordering on thermal stability of lysozyme, α-chymotrypsinogen A, and alcohol dehydrogenase

Effects of water activity (a_W) and solvent ordering were separately analyzed on the thermal unfolding of lysozyme and α-chymotrypsinogen A, and also on the thermal deactivation of yeast alcohol dehydrogenase (YADH) in aqueous solutions with various additives. With the coexistence of additives, water activity was the determinant of the extent of the change in the thermal stability of proteins while solvent ordering was the determinant of the direction of the change. The parameter α, determined from the activity coefficient of water, representing the deviation of a_W from that of the ideal solution, was useful as a quantitative index of the solvent ordering showing good correlations with the unfolding temperature and enthalpy of lysozyme and α-chymotrypsinogen A and also with the thermal deactivation rate constant of YADH at a constant a_W. Solvent ordering seemed to affect the thermal stability of proteins mainly through its effect on the intramolecular hydrophobic interaction among amino acid residues in a protein molecule but the contribution of the electrostatic interaction including hydrogen bonding through the change in permittivity of solution was also suggested.     

Cationic oligopeptides with the repeating sequence L‐lysyl‐L‐alanyl‐L‐alanine: conformational and thermal stability study using optical spectroscopic methods

The infrared (IR), vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) spectra of short cationic sequential peptides (L ‐Lys‐L ‐Ala‐L ‐Ala)_n (n = 1, 2, and 3) were measured over a range of temperatures (20–90 °C) in aqueous solution at near‐neutral pH values in order to investigate their solution conformations and thermally induced conformational changes. VCD spectra of all three oligopeptides measured in the amide I′ region indicate the presence of extended helical polyproline II (PPII)‐like conformation at room temperature. UV‐ECD spectra confirmed this conclusion. Thus, the oligopeptides adopt a PPII‐like conformation, independent of the length of the peptide chain. However, the optimized dihedral angles ? and ψ are within the range ?82 to ?107° and 143–154°, respectively, and differ from the canonical PPII values. At elevated temperatures, the observed intensity and bandshape variations in the VCD and ECD spectra show that the PPII‐like conformation of the Lys‐Ala‐Ala sequence is still preferred, being in equilibrium with an unordered conformer at near‐neutral pH values within the range of temperatures from 20 to 90 °C. This finding was obtained from analysis of the temperature‐dependent spectra using the singular value decomposition method. The study presents KAA‐containing oligopeptides as conformationally stable models of biologically important cationic peptides and proteins. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular dynamic studies on the impact of mutations on the structure,stability, and N‐terminal orientation of annexin A1: Implications for membrane aggregation

Multiple MD simulations were performed for the full‐length wild‐type A1, the full length A1 mutations S27E and S27A, as well as the N‐terminal peptide (AMVSEFLKQAWFIDNEEQEYIKTVKG S ²⁷ KGGPGSAVSPYPTFN) of wild‐type A1 and mutations S27E and S27A. The MD simulation trajectories of about 350 ns were generated and analyzed to examine the changes of core domain calcium binding affinity, core domain and N‐terminal domain structures, and N‐terminal domain orientation. Our results indicated that S27A and S27E mutations caused little changes on the calcium‐binding affinity of the core domain of A1. However, the S27A mutation made the N‐terminal domain of A1 less helical, and made the N‐terminal domain migrate faster toward the core domain; these impacts on A1 are beneficial to the membrane aggregation process. On the contrary, the S27E mutation made the N‐terminal domain of A1 more stable, and hindered the migration to the core domain; these changes on A1 are antagonistic for the membrane aggregation process. Our results using MD simulations provide an atomistic explanation for experimental observations that the S27E mutant showed a higher calcium concentration requirement and lower maximal extent of aggregation, while the wild‐type and two mutants S27E and S27A required identical calcium concentrations for liposome binding. Proteins 2014; 82:3327–3334. © 2014 Wiley Periodicals, Inc.     

NMR studies on thermal stability of α‐helix conformation of melittin in pure ethanol and ethanol–water mixture solvents

Thermal stability of the α‐helix conformation of melittin in pure ethanol and ethanol–water mixture solvents has been investigated by using NMR spectroscopy. With increase in water concentration of the mixture solvents (from 0 wt% to ~71.5 wt%) as well as temperature (from room temperature to 60 °C), the intramolecular hydrogen bonds formed in melittin are destabilized and the α‐helix is partially uncoiled. Further, the hydrogen bonds are found to be more thermally stable in pure ethanol than in pure methanol, suggesting that their stability is enhanced with increase in the size of the alkyl groups of alcohol molecules. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Human Calprotectin： Effect of Calcium and Zinc on its Secondary and Tertiary Structures, and Role of pH in its Thermal Stability

Calprotectin, a heterodimeric complex belonging to the S 100 protein family, has been found predominantly in the cytosolic fraction of neutrophils. In the present study, human calprotectin was purified from neutrophils using two-step ion exchange chromatography. The purified protein was used for circular dichroism study and fluorescence analysis in the presence of calcium and zinc at physiological concentrations, as well as for assessment of its inhibitory activity on the K562 leukemia cell line. The thermal stability of the protein at pH 7.0 （physiological pH） and 8.0 （similar to intestinal pH） was also compared. The results of cell proliferation analysis revealed that human calprotectin initiated growth inhibition of the tumor cells in a dose- dependent manner. The intrinsic fluorescence emission spectra of human calprotectin （50 ktg/ml） in the presence of calcium and zinc ions show a reduction in fluorescence intensity, reflecting a conformational change within the protein with exposure of aromatic residues to the protein surface that is important for the biological function of calprotectin. The far ultraviolet-circular dichroism spectra of human calprotectin in the presence of calcium and zinc ions at physiological concentrations show a decrease in the m-helical content of the protein and an increase in [3- and other structures. Our results also show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of calprotectin in the gut.     

The effects of intramolecular and intermolecular electrostatic repulsions on the stability and aggregation of NISTmAb revealed by HDX‐MS,DSC, and nanoDSF

The stability and aggregation of NIST monoclonal antibody (NISTmAb) were investigated by hydrogen/deuterium exchange mass spectrometry (HDX‐MS), differential scanning calorimetry (DSC), and nano‐differential scanning fluorimetry (nanoDSF). NISTmAb was prepared in eight formulations at four different pHs (pH 5, 6, 7, and 8) in the presence and absence of 150 mM NaCl and analyzed by the three methods. The HDX‐MS results showed that NISTmAb is more conformationally stable at a pH near its isoelectric point (pI) in the presence of NaCl than a pH far from its pI in the absence of NaCl. The stabilization effects were global and not localized. The midpoint temperature of protein thermal unfolding transition results also showed the C_H2 domain of the protein is more conformationally stable at a pH near its pI. On the other hand, the onset of aggregation temperature results showed that NISTmAb is less prone to aggregate at a pH far from its pI, particularly in the absence of NaCl. These seemingly contradicting results, higher conformational stability yet higher aggregation propensity near the pI than far away from the pI, can be explained by intramolecular and intermolecular electrostatic repulsion using Lumry‐Eyring model, which separates folding/unfolding equilibrium and aggregation event. The further a pH from the pI, the higher the net charge of the protein. The higher net charge leads to greater intramolecular and intermolecular electrostatic repulsions. The greater intramolecular electrostatic repulsion destabilizes the protein and the greater intermolecular electrostatic repulsion prevents aggregation of the protein molecules at pH far from the pI.     

Stressor interactions in freshwater habitats: Effects of cold water exposure and food limitation on early‐life growth and upper thermal tolerance in white sturgeon,Acipenser transmontanus

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Influence of the H‐site residue 108 on human glutathione transferase P1‐1 ligand binding: Structure‐thermodynamic relationships and thermal stability

The effect of the Y108V mutation of human glutathione S‐transferase P1‐1 (hGST P1‐1) on the binding of the diuretic drug ethacrynic acid (EA) and its glutathione conjugate (EASG) was investigated by calorimetric, spectrofluorimetric, and crystallographic studies. The mutation Tyr 108 → Val resulted in a 3D‐structure very similar to the wild type (wt) enzyme, where both the hydrophobic ligand binding site (H‐site) and glutathione binding site (G‐site) are unchanged except for the mutation itself. However, due to a slight increase in the hydrophobicity of the H‐site, as a consequence of the mutation, an increase in the entropy was observed. The Y108V mutation does not affect the affinity of EASG for the enzyme, which has a higher affinity (K_d ～ 0.5 μM) when compared with those of the parent compounds, K ～ 13 μM, K ～ 25 μM. The EA moiety of the conjugate binds in the H‐site of Y108V mutant in a fashion completely different to those observed in the crystal structures of the EA or EASG wt complex structures. We further demonstrate that the ΔC_p values of binding can also be correlated with the potential stacking interactions between ligand and residues located in the binding sites as predicted from crystal structures. Moreover, the mutation does not significantly affect the global stability of the enzyme. Our results demonstrate that calorimetric measurements maybe useful in determining the preference of binding (the binding mode) for a drug to a specific site of the enzyme, even in the absence of structural information.     

Effect of neohesperidin dihydrochalcone on the activity and stability of alpha‐amylase: a comparative study on bacterial,fungal, and mammalian enzymes

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha‐amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha‐amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha‐amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha‐amylase surface in domain B. This domain shows differences in various alpha‐amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact. Copyright © 2015 John Wiley & Sons, Ltd.     

FTIR spectroscopic studies on aggregation process of the β‐amyloid 11–28 fragment and its variants

Aggregation of Aβ peptides is a seminal event in Alzheimer's disease. Detailed understanding of the Aβ assembly process would facilitate the targeting and design of fibrillogenesis inhibitors. Here, conformational studies using FTIR spectroscopy are presented. As a model peptide, the 11–28 fragment of Aβ was used. This model peptide is known to contain the core region responsible for Aβ aggregation. The structural behavior of the peptide during aggregation provoked by the addition of water to Aβ(11–28) solution in hexafluoroisopropanol was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21–23 (A21G, E22K, E22G, E22Q and D23N). The results showed that the aggregation of the peptides proceeds via a helical intermediate, and it is possible that the formation of α‐helical structures is preceded by creation of 3₁₀‐helix/3₁₀‐turn structures. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Computationally‐predicted CB1 cannabinoid receptor mutants show distinct patterns of salt‐bridges that correlate with their level of constitutive activity reflected in G protein coupling levels,thermal stability,and ligand binding

The cannabinoid receptor 1 (CB1), a member of the class A G‐protein‐coupled receptor (GPCR) family, possesses an observable level of constitutive activity. Its activation mechanism, however, has yet to be elucidated. Previously we discovered dramatic changes in CB1 activity due to single mutations; T3.46A, which made the receptor inactive, and T3.46I and L3.43A, which made it essentially fully constitutively active. Our subsequent prediction of the structures of these mutant receptors indicated that these changes in activity are explained in terms of the pattern of salt‐bridges in the receptor region involving transmembrane domains 2, 3, 5, and 6. Here we identified key salt‐bridges, R2.37 + D6.30 and D2.63 + K3.28, critical for CB1 inactive and active states, respectively, and generated new mutant receptors that we predicted would change CB1 activity by either precluding or promoting these interactions. We find that breaking the R2.37 + D6.30 salt‐bridge resulted in substantial increase in G‐protein coupling activity and reduced thermal stability relative to the wild‐type reflecting the changes in constitutive activity from inactive to active. In contrast, breaking the D2.63 + K3.28 salt‐bridge produced the opposite profile suggesting this interaction is critical for the receptor activation. Thus, we demonstrate an excellent correlation with the predicted pattern of key salt‐bridges and experimental levels of activity and conformational flexibility. These results are also consistent with the extended ternary complex model with respect to shifts in agonist and inverse agonist affinity and provide a powerful framework for understanding the molecular basis for the multiple stages of CB1 activation and that of other GPCRs in general. Proteins 2013; 81:1304–1317. © 2013 Wiley Periodicals, Inc.     

Effects of N2,N2-dimethylguanosine on RNA structure and stability: crystal structure of an RNA duplex with tandem m2 2G:A pairs

Methylation of the exocyclic amino group of guanine is a relatively common modification in rRNA and tRNA. Single methylation (N²-methylguanosine, m²G) is the second most frequently encountered nucleoside analog in Escherichia coli rRNAs. The most prominent case of dual methylation (N²,N²-dimethylguanosine, m²₂G) is found in the majority of eukaryotic tRNAs at base pair m²₂G26:A44. The latter modification eliminates the ability of the N² function to donate in hydrogen bonds and alters its pairing behavior, notably vis-à-vis C. Perhaps a less obvious consequence of the N²,N²-dimethyl modification is its role in controlling the pairing modes between G and A. We have determined the crystal structure of a 13-mer RNA duplex with central tandem m²₂G:A pairs. In the structure both pairs adopt an imino-hydrogen bonded, pseudo-Watson–Crick conformation. Thus, the sheared conformation frequently seen in tandem G:A pairs is avoided due to a potential steric clash between an N²-methyl group and the major groove edge of A. Additionally, for a series of G:A containing self-complementary RNAs we investigated how methylation affects competitive hairpin versus duplex formation based on UV melting profile analysis.     

A study on body temperature regulation and residential thermal environments of the elderly—I. whole country survey of residential thermal environment and evaluation methods: RTE-index

1. 1. As part of “research on environmental comfort,” that is, research which aims to make people's living environment more comfortable, we attempted to clarify the relation between the living environment of elderly people and physiological and psychological factors. We carried out a comprehensive study with a view to establishing comfort standards for the residential thermal environment, and for creating evaluation and control systems.

2. 2. The comfort of the living environment is closely related to the thermal environment and the temperature-regulation ability of the human body. This ability of the body to regulate temperature develops during childhood and recedes as the adult ages.

3. 3. We therefore carried out: (a) experiments on body-temperature regulation and on special characteristics of the body-temperature regulation of elderly people, (b) a nationwide survey of the actual residential thermal environments of elderly people and (c) a survey of the daily activities of elderly people, and how elderly people's physiological and psychological conditions change when they are engaged in these activities.

4. 4. As a result of these experiments and surveys, we were able to: (d) formulate standards for evaluation of the residential thermal environment and (e) numerically express the results of systematic evaluation of residential thermal environments of elderly people, by means of a RTE-index.

Substitution of a proline for alanine 183 in the hinge region of phosphoglycerate kinase: Effects on catalysis, activation by sulfate, and thermal stability

A hinge-bending domain movement has been postulated as an important part of the catalytic mechanism of phosphoglycerate kinase (PGK) (Bankset al., 1979). In order to test the role of the flexibility of a putative interdomain hinge in the substrate- and sulfate-induced conformational transitions, alanine-183 was replaced by proline using site-directed mutagenesis. The maximal velocity of the Ala 183Pro mutant, measured at saturating concentrations of ATP and phosphoglycerate (5 mM and 10 mM, respectively) and in the absence of sulfate ions, is increased approximately 21% in comparison to the wild type PGK. TheK _m values for both substrates are essentially unchanged. The effect of sulfate on the specific activity of the Ala 183Pro mutant and the wild type PGK was measured in the presence of 1 mM ATP and 2 mM 3-phosphoglycerate (3-PG). A maximum activation of 70% was observed at 20 mM sulfate for the mutant enzyme, as compared to 130% activation at 30 mM sulfate for the wild type PGK. These results demonstrate that the increased rigidity of the putative hinge, introduced by the AlaPro mutation, does not impair catalytic efficiency of phosphoglycerate kinase, while it appears to decrease the sulfate-dependent activation. The differential scanning calorimetry (DSC) studies demonstrate an increased susceptibility of the Ala 183 Pro mutant to thermal denaturation. In contrast to one asymmetric transition observed in the DSC scan for the wild type PGK, withT _m near 54°C, two transitions are evident for the mutant enzyme withT _m values of about 45 and 54°C. Using a thermodynamic model for two interacting domains, a decrease in the free energy of domain-domain interactions of about 2 kcal was estimated from the DSC data.     

Biogenesis,characterization, and the effect of vicenin‐gold nanoparticles on glucose utilization in 3T3‐L1 adipocytes: A bioinformatic approach to illuminate its interaction with PTP 1B and AMPK

This study reported the synthesis of Vicenin‐2 gold nanoparticles (VN‐AuNPs) and evaluated their effect on the glucose utilization efficiency of 3T3‐L1 adipocytes. The VN‐AuNPs were characterized by microscopic, DLS and spectral analysis. The bio‐reducing efficiency of Vicenin‐2 (VN) was computed and confirmed by HPLC analysis. The stability of VN‐AuNPs in various physiological media was explored. The cytotoxicity and glucose uptake assays were performed in 3T3‐L1 adipocytes. The docking of VN with PTP1B and AMPK was also performed. The color change and UV absorption at 537 nm preliminarily confirmed the VN reduced gold nanoparticles. The VN‐AuNPs appeared as spherical particles (57 nm) and face centered cubic crystals under TEM and XRD analysis, respectively. Its zeta potential was found to be ?6.53 mV. The FT‐IR spectra of VN and its AuNPs confirmed its stability. The computed reducing potential of VN was similar to the extent of VN utilized during the synthesis of VN‐AuNPs. The VN‐AuNPs showed a remarkable stability in different physiological media. At 100 µM concentration, VN‐AuNPs displayed 78.21% cell viability. A concentration dependent increase in glucose uptake was noted in 3T3‐L1 adipocytes when incubated with VN‐AuNPs. The docking data revealed a strong interaction of VN with the binding pockets of PTP1B and AMPK. This demonstrates that the fabricated VN‐AuNPs might enhance the intracellular VN availability mediated cellular glucose utilization and this would serve as a novel nanodrug for the management of diabetes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1096–1106, 2015     

Further studies on cell adhesion based on Lex-Lex interaction,with new approaches: embryoglycan aggregation of F9 teratocarcinoma cells,and adhesion of various tumour cells based on Lex expression

We previously proposed specific interaction of Le^x (Gal1 4[Fuc1 3]-GlcNAc1 3Gal) with Le^x as a basis of cell adhesion in pre-implantation embryos and in aggregation of F9 teratocarcinoma cells, based on several lines of evidence (Eggenset al., J Biol Chem (1989)264:9476–9484). We now present additional evidence for this concept, based on autoaggregation studies of plastic beads coated with glycosphingolipids (GSLs) bearing Le^x or other epitopes, and affinity chromatography on Le^x-columns of multivalent lactofucopentaose III (Le^x oligosaccharide) conjugated with lysyllysine. Comparative adhesion studies of Le^x-expressing tumour cellsvs their Le^x-non-expressing variants showed that only Le^x-expressing cells adhere to Le^x-coated plates and are involved in tumour cell aggregation, in analogy to F9 cell aggregation. The major carrier of Le^x determinant in F9 cells is not GSL but rather polylactosaminoglycan (embryoglycan), and we demonstrated autoaggregation of purified embryoglycan in the presence of Ca²⁺, and reversible dissociation in the absence of Ca²⁺ (addition of EDTA). Defucosylated embryoglycan did not show autoaggregation under the same conditions. Thus, Le^x-Le^x interaction has been demonstrated on a lactosaminoglycan basis as well as a GSL basis. A molecular model of Le^x-Le^x interaction based on minimum energy conformation with involvement of Ca²⁺ is presented.Abbreviations BSA bovine serum albumin - CHO carbohydrate - DMEM Dulbecco's modified Eagle's medium - EDTA ethylenediaminetetraacetic acid - GP glycopeptide - GSL glycosphingolipid - LAG lactosaminoglycan - Le^x Gal1 4[Fuc-1 3]GlcNAc1 R - LFP lacto-N-fucopentaose - LysLys-OH lysyllysinol - Mr relative molecular weight - PBS phosphate-buffered saline - PG paragloboside (Gal1 4GlcNAc1 3Gal1 4Glc1 1Cer) - TBS Tris-buffered saline (10mM Tris-HCl, pH 7.4, containing 0.15M NaCl) - TC tumour cell     

An eco‐friendly direct spectrofluorimetric method for the determination of irreversible tyrosine kinase inhibitors,neratinib and pelitinib: application to stability studies

A new rapid and simple stability‐indicating spectrofluorimetric method has been developed for the determination of two irreversible tyrosine kinase inhibitors (TKIs), neratinib (NER) and pelitinib (PEL). The method is based upon measurement of the native fluorescence intensity of both drugs at λ_ex 270 nm in aqueous borate buffer solutions (pH 10.5). The fluorescence intensity recorded at 545 nm (NER) and 465 nm (PEL) were rectilinear over the concentration range of 0.1–10 μg/mL for both drugs with a high correlation coefficient (r > 0.999). The proposed method provided low limits of detection and of quantitation of 0.07, 0.11 μg/mL (NER) and 0.02, 0.05 μg/mL (PEL), respectively. The method was successfully applied for the determination of NER and PEL in bulk powder. The proposed methods were fully validated as per the International Conference on Harmonisation (ICH) guidelines. The application of the method was extended to stability studies of both NER and PEL under different forced‐degradation conditions (acidic‐induced, base‐induced, oxidative, wet heat, and photolytic degradation). Moreover, the kinetics of the base‐induced and oxidative degradation of both drugs was investigated and the pseudo‐first‐order rate constants and half‐lives were estimated at different temperatures. Also, an Arrhenius plot was applied to predict the stability behaviour of the two drugs at room temperature. Copyright © 2016 John Wiley & Sons, Ltd.