On-line chemiluminescence determination of mitoxantrone by capillary electrophoresis

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for the determination of mitoxantrone (MTX) has been developed, which based on the CL reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by MTX. Under optimum analytical conditions, MTX is determined over the range of 7.0 × 10⁻⁸–1.0 × 10⁻⁶ M with a detection limit of 1.0 × 10⁻⁸ M. The relative standard deviation (RSD) was 3.7%, 2.6% and 3.0% for 7.0 × 10⁻⁸, 5.0 × 10⁻⁷ and 1.0 × 10⁻⁶ M MTX (n = 11), respectively. In laboratory-built CE–CL apparatus, the proposed method has been applied to determination of MTX in commercial drug and spiked in human urine and plasma with satisfactory results.     

Chemiluminescent determination of H2O2 using 4-(1,2,4-triazol-1-yl)phenol as an enhancer based on the immobilization of horseradish peroxidase onto magnetic beads

This article describes the employment of a novel p-phenol derivative, 4-(1,2,4-triazol-1-yl)phenol (TRP), as a highly potent signal enhancer of the luminol-hydrogen peroxide (H₂O₂)-horseradish peroxidase (HRP) chemiluminescence (CL) system. The CL reaction conditions were optimized, and the enhancement characteristics of TRP were compared with those of p-iodophenol (PIP). TRP produced a strong enhancement of the CL with the effect of prolonging the light emission. The developed system was then applied to the determination of H₂O₂ with immobilized HRP using magnetic beads as a solid support. The linear range for H₂O₂ was 2.0 × 10⁻⁶ to 1.0 × 10⁻³ M. The detection limit for H₂O₂ was 2.0 × 10⁻⁶ M. The proposed sensor was applied successfully to the determination of H₂O₂ in rainwater.     

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.     

Quantification of carnosine-related peptides by microchip electrophoresis with chemiluminescence detection

A microchip electrophoresis (MCE) method with chemiluminescence (CL) detection was developed for the determination of carnosine-related peptides, including carnosine, homocarnosine, and anserine, in biological samples. A simple integrated MCE-CL system was built to perform the assays. The highly sensitive CL detection was achieved by means of the CL reaction between hydrogen peroxide and N-(4-aminobutyl)-N-ethylisoluminol-tagged peptides in the presence of adenine as a CL enhancer and Co²⁺ as a catalyst. Experimental conditions for analyte labeling, MCE separation, and CL detection were studied. MCE separation of the above-mentioned three peptides took less than 120 s. Detection limits (signal/noise ratio [S/N] = 3) of 3.0 × 10⁻⁸, 2.8 × 10⁻⁸, and 3.4 × 10⁻⁸ M were obtained for carnosine, anserine, and homocarnosine, respectively. The current MCE-CL method was applied for the determination of carnosine, anserine, and homocarnosine in human cerebrospinal fluid (CSF) and canine plasma. Homocarnosine was detected at the micromolar (μM) level in the CSF samples analyzed, whereas the levels of carnosine and anserine in these samples were below the detection limit of the assay. Interestingly, both carnosine and anserine were detected in the canine plasma samples, whereas homocarnosine was not.     

Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea and guanidine hydrochloride unfolding

Low concentrations of urea and GuHCl (2 M) enhanced the activity of endoglucanase (EC 3.1.2.4) from Aspergillus aculeatus by 2.3- and 1.9-fold, respectively. The K_m values for controls, in the presence of 2 M urea and GuHCl, were found to be 2.4 ± 0.2 × 10⁻⁸ mol L⁻¹, 1.4 ± 0.2 × 10⁻⁸ mol L⁻¹, and 1.6 ± 0.2 × 10⁻⁸ mol L⁻¹, respectively. The dissociation constant (K_d) showed changes in the affinity of the enzyme for the substrate with increases in the K_cat suggesting an increased turnover number in the presence of urea and GuHCl. Fluorescence studies showed changes in the microenvironment of the protein. The increase in the activity of this intermediate state was due to conformational changes accompanied by increased flexibility at the active site.     

Xanthine microsensor based on polypyrrole molecularly imprinted film modified carbon fiber microelectrodes

A molecularly imprinted polymers (MIPs) microsensor was presented as a carbon fiber microelectrode (CFME) coating for specifically recognizing xanthine (Xan). The polymeric film was obtained based on the imprinted procedure of electropolymerization of pyrrole in the presence of the template molecule Xan by cyclic voltammetry, and template was removed by magnetic stirring. Under the optimum conditions, a satisfactory molecularly binding selectivity of Xan was obtained from the MIPs microsensor with an imprinting factor (IF) of 6.63 and a linear response to concentration in certain ranges. The ranges are from 4.0 × 10⁻⁶ to 6.0 × 10⁻⁵ M and from 8.0 × 10⁻⁵ to 2.0 × 10⁻³ M with a detection limit of 2.5 × 10⁻⁷ M. Meanwhile, good stability (relative standard deviation [RSD] = 3.2%, n = 10) and reproducibility (RSD = 2.0%, n = 10) were observed, and recoveries ranging from 96.9 to 102.5% were calculated when applied to Xan determination in real blood serum samples.     

Biosensor based on the biocatalysis of microperoxidase-11 in nanocomposite material of multiwalled carbon nanotubes/room temperature ionic liquid for amperometric determination of hydrogen peroxide

A novel nanocomposite material of multiwalled carbon nanotubes (MWCNTs) and room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF₆) was explored and used to construct a novel microperoxidase-11 (MP-11) biosensor for the determination of hydrogen peroxide (H₂O₂). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to characterize the performance of the biosensor. Under the optimized experimental conditions, H₂O₂ could be detected in a linear calibration range of 0.5 to 7.0 × 10⁻⁷ mol L⁻¹ with a correlation coefficient of 0.9949 (n = 9) and a detection limit of 3.8 × 10⁻⁹ mol L⁻¹ at 3σ. The modified electrodes displayed excellent electrochemical response, high sensitivity, long-term stability, and good bioactivity and selectivity.     

Ibuprofen modulates allosterically NO dissociation from ferrous nitrosylated human serum heme-albumin by binding to three sites

Human serum albumin (HSA) is a monomeric allosteric protein. Here, the effect of ibuprofen on denitrosylation kinetics (k_off) and spectroscopic properties of HSA-heme-Fe(II)-NO is reported. The k_off value increases from (1.4 ± 0.2) × 10⁻⁴ s⁻¹, in the absence of the drug, to (9.5 ± 1.2) × 10⁻³ s⁻¹, in the presence of 1.0 × 10⁻² M ibuprofen, at pH 7.0 and 10.0 °C. From the dependence of k_off on the drug concentration, values of the dissociation equilibrium constants for ibuprofen binding to HSA-heme-Fe(II)-NO (K₁ = (3.1 ± 0.4) × 10⁻⁷ M, K₂ = (1.7 ± 0.2) × 10⁻⁴ M, and K₃ = (2.2 ± 0.2) × 10⁻³ M) were determined. The K₃ value corresponds to the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(II)-NO determined by monitoring drug-dependent absorbance spectroscopic changes (H = (2.6 ± 0.3) × 10⁻³ M). Present data indicate that ibuprofen binds to the FA3-FA4 cleft (Sudlow’s site II), to the FA6 site, and possibly to the FA2 pocket, inducing the hexa-coordination of HSA-heme-Fe(II)-NO and triggering the heme-ligand dissociation kinetics.     

Estrogenic activities of Psoralea corylifolia L. seed extracts and main constituents

Estrogenic activities of ethanol extract and its active components from Psoralea corylifolia L. were studied using various in vitro assays. The main components from ethanol extract were analyzed to be bakuchiol, psoralen, isobavachalcone, isobavachromene, and bavachinin. In a fractionation procedure, hexane and chloroform fractions showed estrogenic activity in yeast transactivation assay and E-screen assay. In yeast transactivation assay, ethanol extract, hexane, and chloroform fractions showed significantly higher activities at a concentration of 1.0 ng/ml, and bakuchiol at the concentration of 10⁻⁶ M was showed the highest activity, especially, which was higher than genistein at the same concentration. In E-screen assay, cell proliferation of bakuchiol (10⁻⁶ M) showed similar estrogenic activity with genistein (10⁻⁶ M). In ER binding assay, bakuchiol displayed the strongest ER-binding affinity (IC₅₀ for ERα = 1.01 × 10⁻⁶ M, IC₅₀ for ERβ = 1.20 × 10⁻⁶ M) and bakuchiol showed five times higher affinity for ERα than for ERβ.     

Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell’s osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V_b) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and −38.1 ± 1.2%. The permeability of the oocytes to water (L_p) was determined to be 3.8 ± 0.4 × 10⁻², 5.7 ± 0.8 × 10⁻², and 13.2 ± 1.3 × 10⁻² μm min⁻¹ atm⁻¹, when measured at temperatures of 5, 10 and 20 °C. The respective EG permeability values (P_s) were 9.5 ± 0.1 × 10⁻⁵, 14.6 ± 1.2 × 10⁻⁵, and 41.7 ± 2.4 × 10⁻⁵ cm min⁻¹. The activation energies for L_p and P_s were determined to be 14.5 and 17.5 kcal mol⁻¹, respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 °C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.     

Inhibition of the cardiac inward rectifier potassium currents by KB-R7943

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea) was developed as a specific inhibitor of the sarcolemmal sodium–calcium exchanger (NCX) with potential experimental and therapeutic use. However, KB-R7943 is shown to be a potent blocker of several ion currents including inward and delayed rectifier K⁺ currents of cardiomyocytes. To further characterize KB-R7943 as a blocker of the cardiac inward rectifiers we compared KB-R7943 sensitivity of the background inward rectifier (I_K1) and the carbacholine-induced inward rectifier (I_KACh) currents in mammalian (Rattus norvegicus; rat) and fish (Carassius carassius; crucian carp) cardiac myocytes. The basal I_K1 of ventricular myocytes was blocked with apparent IC50-values of 4.6 × 10^− 6 M and 3.5 × 10^− 6 M for rat and fish, respectively. I_KACh was almost an order of magnitude more sensitive to KB-R7943 than I_K1 with IC₅₀-values of 6.2 × 10^− 7 M for rat and 2.5 × 10^− 7 M for fish. The fish cardiac NCX current was half-maximally blocked at the concentration of 1.9–3 × 10^− 6 M in both forward and reversed mode of operation. Thus, the sensitivity of three cardiac currents to KB-R7943 block increases in the order I_K1 ~ I_NCX < I_KACh. Therefore, the ability of KB-R7943 to block inward rectifier potassium currents, in particular I_KACh, should be taken into account when interpreting the data with this inhibitor from in vivo and in vitro experiments in both mammalian and fish models.     

Kinetic and thermodynamic properties of the folding and assembly of formate dehydrogenase

The folding mechanism and stability of dimeric formate dehydrogenase from Candida methylica was analysed by exposure to denaturing agents and to heat. Equilibrium denaturation data yielded a dissociation constant of about 10⁻¹³ M for assembly of the protein from unfolded chains and the kinetics of refolding and unfolding revealed that the overall process comprises two steps. In the first step a marginally stable folded monomeric state is formed at a rate (k₁) of about 2 × 10⁻³ s⁻¹ (by deduction k₋₁ is about10⁻⁴ s⁻¹) and assembles into the active dimeric state with a bimolecular rate constant (k₂) of about 2 × 10⁴ M⁻¹ s⁻¹. The rate of dissociation of the dimeric state in physiological conditions is extremely slow (k₋₂ ∼ 3 × 10⁻⁷ s⁻¹).     

Characterization of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase multigene family of Malus domestica Borkh

Two 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes have been cloned from RNA isolated from leaf tissue of apple (Malus domestica cv. Royal Gala). The genes, designated MD-ACO2 (with an ORF of 990 bp) and MD-ACO3 (966 bp) have been compared with a previously cloned gene of apple, MD-ACO1 (with an ORF of 942 bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9% in the ORF but diverge in the 3′ untranslated regions (3′-UTR) (69.5%). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5%) and MD-ACO2 (77.8%) in the ORF, and 68.4% (MD-ACO1) and 71% (MD-ACO2) in the 3′-UTR. Southern analysis confirmed that MD-ACO3 is encoded by a distinct gene, but the distinction between MD-ACO1 and MD-ACO2 is not as definitive. Gene expression analysis has shown that MD-ACO1 is restricted to fruit tissues, with optimal expression in ripening fruit, MD-ACO2 expression occurs more predominantly in younger fruit tissue, with some expression in young leaf tissue, while MD-ACO3 is expressed predominantly in young and mature leaf tissue, with less expression in young fruit tissue and least expression in ripening fruit. Protein accumulation studies using western analysis with specific antibodies raised to recombinant MD-ACO1 and MD-ACO3 produced in E. coli confirmed the accumulation of MD-ACO1 in mature fruit, and an absence of accumulation in leaf tissue. In contrast, MD-ACO3 accumulation occurred in younger leaf tissue, and in younger fruit tissue. Further, the expression of MD-ACO3 and accumulation of MD-ACO3 in leaf tissue is linked to fruit longevity. Analysis of the kinetic properties of the three apple ACOs using recombinant enzymes produced in E. coli revealed apparent Michaelis constants (K_m) of 89.39 μM (MD-ACO1), 401.03 μM (MD-ACO2) and 244.5 μM (MD-ACO3) for the substrate ACC, catalytic constants (K_cat) of 6.6 × 10⁻² (MD-ACO1), 3.44 × 10⁻² (Md-ACO2) and 9.14 × 10⁻² (MD-ACO3) and K_cat/K_m (μM s⁻¹) values of 7.38 × 10⁻⁴ μM s⁻¹ (MD-ACO1), 0.86 × 10⁻⁴ M s⁻¹ (MD-ACO2) and 3.8 × 10⁻⁴ μM s⁻¹ (MD-ACO3). These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed in apple fruit and leaf tissue, an expression pattern that is supported by some variation in kinetic properties.     

Benzo[a]pyrene degradation using simultaneously combined chemical oxidation,biotreatment with Fusarium solani and cyclodextrins

The interest of simultaneously combining chemical (Fenton’s reaction) and biological treatments for the degradation of a high molecular weight polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) has been studied in laboratory tests. An optimal concentration of 1.5 × 10⁻³ M H₂O₂ as Fenton’s reagent was firstly determined as being compatible with the growth of Fusarium solani, the Deuteromycete fungus used in the biodegradation process. For the enhancement of BaP solubilisation, cyclodextrins were also used in the performed tests. The best degradation performance was achieved through the use of 5 × 10⁻³ M hydroxypropyl-β-cyclodextrin (HPBCD) in comparison with randomly methylated-β-cyclodextrin (RAMEB). When Fenton’s treatment was combined with biodegradation, a beneficial effect on BaP degradation (25%) was obtained in comparison with biodegradation alone (8%) or with chemical oxidation alone (16%) in the presence of HPBCD for 12 days of incubation.     

The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2

Peroxiredoxin 2 (Prx2) is a 2-Cys peroxiredoxin extremely abundant in the erythrocyte. The peroxidase activity was studied in a steady-state approach yielding an apparent K_M of 2.4 μM for human thioredoxin and a very low K_M for H₂O₂ (?0.7 μM). Rate constants for the reaction of peroxidatic cysteine with the peroxide substrate, H₂O₂ or peroxynitrite, were determined by competition kinetics, k₂ = 1.0 × 10⁸ and 1.4 × 10⁷ M⁻¹ s⁻¹ at 25 °C and pH 7.4, respectively. Excess of both oxidants inactivated the enzyme by overoxidation and also tyrosine nitration and dityrosine were observed with peroxynitrite treatment. Prx2 associates into decamers (5 homodimers) and we estimated a dissociation constant K_d < 10⁻²³ M⁴ which confirms the enzyme exists as a decamer in vivo. Our kinetic results indicate Prx2 is a key antioxidant enzyme for the erythrocyte and reveal red blood cells as active oxidant scrubbers in the bloodstream.     

Single-strand DNA translation initiation step analyzed by Isothermal Titration Calorimetry

Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K_d = 3.62 ± 2.1 × 10⁻⁸ M) or the RNA corresponding sequence (K_d = 2.7 ± 0.82 × 10⁻⁸ M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.     

Accumulation of lead by free and immobilized cyanobacteria with special reference to accumulation factor and recovery

Lead accumulation by free and immobilized cyanobacteria, Lyngbya majuscula and Spirulina subsalsa was studied. Exponentially growing biomass was exposed to 1-20 mg L⁻¹ of Pb(II) solution at pH 6, 7 and 8 for time periods ranging from 10 min to 48 h. L. majuscula accumulated 10 times more Pb (13.5 mg g⁻¹) than S. subsalsa (1.32 mg g⁻¹) at pH 6 within 3 h of exposure to 20 mg L⁻¹ Pb(II) solution and 76% of the Pb could be recovered using 0.1 M EDTA. This chelator (2 μM) did not influence Pb accumulation whereas 100 μM citrate increased that of S. subsalsa 6- to 8-fold. L. majuscula filaments enmeshed in a glass wool packed in a column removed 95.8% of the Pb from a 5 mg L⁻¹ Pb solution compared to free and dead biomass which removed 64 and 33.6% Pb respectively. A 92.5% recovery of accumulated Pb from the immobilized biomass suggests that repeated absorption-desorption is possible.     

Facile synthesis of fluorescent polysaccharides: Cytosine grafted agarose and κ-carrageenan

New fluorescent polymeric materials were synthesized by grafting the nucleobase cytosine on to the backbone of agarose and κ-carrageenan, employing a rapid water based method under microwave irradiation using potassium persulphate (KPS) as an initiator. The emission spectrum of the modified agarose and κ-carrageenan recorded in aqueous solution (5 × 10⁻⁵ M) exhibited emission maxima (λ_em,max) at 348 nm by excitation at 266 nm. The emission intensity was enhanced by ca. 104% and 60% compared to that of pure cytosine solution of the same concentration. When the concentration of the pure cytosine solution is made equivalent to the concentration of the cytosine molar component (3.09 × 10⁻⁵) and (3.5 × 10⁻⁵) present in 5 × 10⁻⁵ M solution of modified agarose and κ-carrageenan, respectively, then ca. 143% and 81% enhancement in emission intensity was observed. The remarkable fluorescent activity of the agarose-cytosine derivative may have potential uses as sensor in various applications.     

Aqueous solution chemistry of dichloro[S-methylcysteine(N,S)]platinum(II) isomers and their hydrolysis products

Rate and equilibrium constants at 25 °C, pH ∼ 1, and ionic strength 0.10 for hydrolysis of the two non-equivalent chlorides of dichloro[S-methyl-l-cysteine(N,S)]platinum(II) isomers, denoted [PtCl₂(SmecysH)], and the resultant chloro-aqua species have been determined by NMR, potentiometric, and spectrophotometric methods. Though hydrolysis constants, K_h, for the two chlorides are similar (pK_h = 4-5), the rate of hydrolysis of the chloride trans to coordinated S, k_h = 3.4 × 10⁻³ s⁻¹, is 2-3 orders of magnitude faster than the k_h for the other chloride, 2.3 × 10⁻⁶ s⁻¹, and for the cancer drug cisplatin, cis-[PtCl₂(NH₃)₂], 5.2 × 10⁻⁵ s⁻¹. Relative rates of hydrolysis determined under three different experimental conditions (pH ∼ 1 in 0.10 M HNO₃, high pH in 0.10 M NaOH, and at low pH with Ag⁺ assistance) are consistent: the Cl trans to S is 100-1000 times more labile than the Cl cis to S. Potentiometric and NMR methods were also used to estimate pK_a values of all aqua species, which are comparable to values reported for corresponding aqua species derived from cisplatin.     

The kinetics of the hydrogen/deuterium exchange of epidermal growth factor receptor ligands

Five highly homologous epidermal growth factor receptor ligands were studied by mass spectral analysis, hydrogen/deuterium (H/D) exchange via attenuated total reflectance Fourier transform-infrared spectroscopy, and two-dimensional correlation analysis. These studies were performed to determine the order of events during the exchange process, the extent of H/D exchange, and associated kinetics of exchange for a comparative analysis of these ligands. Furthermore, the secondary structure composition of amphiregulin (AR) and heparin-binding-epidermal growth factor (HB-EGF) was determined. All ligands were found to have similar contributions of 3₁₀-helix and random coil with varying contributions of β-sheets and β-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor-α (TGF-α), AR, HB-EGF, and epiregulin (ER), respectively. The rate constants were determined and classified as fast, intermediate, and slow: for EGF the 0.20 min⁻¹ (Tyr), 0.09 min⁻¹ (Arg, β-turns), and 1.88 × 10⁻³ min⁻¹ (β-sheets and 3₁₀-helix); and for TGF-α 0.91 min⁻¹ (Tyr), 0.27 min⁻¹ (Arg, β-turns), and 1.41 × 10⁻⁴ min⁻¹ (β-sheets). The time constants for AR 0.47 min⁻¹ (Tyr), 0.04 min⁻¹ (Arg), and 1.00 × 10⁻⁴ min⁻¹ (buried 3₁₀-helix, β-turns, and β-sheets); for HB-EGF 0.89 min⁻¹ (Tyr), 0.14 min⁻¹ (Arg and 3₁₀-helix), and 1.00 × 10⁻³ min⁻¹ (buried 3₁₀-helix, β-sheets, and β-turns); and for epiregulin 0.16 min⁻¹ (Tyr), 0.03 min⁻¹ (Arg), and 1.00 × 10⁻⁴ min⁻¹ (3₁₀-helix and β-sheets). These results provide essential information toward understanding secondary structure, H/D exchange kinetics, and solvation of these epidermal growth factor receptor ligands in their unbound state.