Fluorescence study on site-specific binding of perfluoroalkyl acids to human serum albumin

Binding of five perfluoroalkyl acids with human serum albumin (HSA) was investigated by site-specific fluorescence. Intrinsic fluorescence of tryptophan-214 in HSA was monitored upon addition of the chemicals. Although perfluorobutyl acid (PFBA) and perfluorobutane sulfonate (PFBS) did not cause fluorescence change, perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorododecanoic acid (PFDoA) induced fluorescence quenching, from which binding constant of 2.7 × 10⁵ M⁻¹ for PFOA and 2.2 × 10⁴ M⁻¹ for PFOS was calculated. Two fluorescent probes, dansylamide (DA) and dansyl-l-proline (DP), were employed in fluorescence displacement measurements to study the interaction at two Sudlow’s binding sites. At Site I, both PFBA and PFBS displaced DA with binding constants of 1.0 × 10⁶ M⁻¹ and 2.2 × 10⁶ M⁻¹. At Site II, PFBS and PFDoA displaced DP with binding constants of 6.5 × 10⁶ M⁻¹ and 1.2 × 10⁶ M⁻¹, whereas PFBA did not bind. The data were compared with fatty acids to evaluate the potential toxicological effect of these environmental chemicals. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reversible binding of tolmetin,zomepirac, and their glucuronide conjugates to human serum albumin and plasma

Acyl glucuronides of drugs and bilirubin have been shown in the past decade to be reactive metabolites undergoing acyl migration and irreversible binding. The latter reaction has been hypothesized to be facilitated by or to proceed through the formation of a reversible complex. Furthermore, it has been suggested that the decreased binding seen in patients with compromised excretory function may be due to competition by elevated plasma concentrations of the glucuronides. In these reversible binding studies, we characterized the extent and the “site” of binding of tolmetin, zomepirac, their glucuronides and isomeric conjugates. We also examined the displacement between the parent drugs and their glucuronide conjugates using a rapid ultrafiltration method. Tolmetin exhibited three classes of binding sites with a primary association constant of 1.7×10⁶ M⁻¹ (K_dl=0.60 μM). The primary association constant of zomepirac (1.16×10⁶ M⁻¹, K_dl=0.86 μM) is similar to that of tolmetin. The β 1 and α/β3 glucuronides of both compounds bind to a lesser extent than their parent aglycones. The isomeric glucuronide conjugates of both compounds showed much stronger binding than the β/1 conjugates. Of the four glucuronides investigated, tolmetin glucuronide-α/β3 isomer was bound by fatty acid free human serum albumin with the highest affinity (4.6×10⁵ M⁻¹, K_d=2.22 μM). Protein binding of the parent drugs and conjugates were decreased significantly at pH 5.0. In displacement studies, except for salicylate and acetylsalicylate, drugs known to bind to Sites I and II as well as the digitoxin and tamoxifen binding sites had little inhibitory effect on the binding of tolmetin, zomepirac, and their glucuronide conjugates. Supported in part by Grant GM 36633 from the National Institute of General Medical Sciences.     

Purification and Characterisation of a Newly Isolated Stable Long-Life Tannase produced by F. subglutinans (Wollenweber and Reinking) Nelson et al.

A stable long-life tannase was synthesised by Fusarium subglutinans and the fermentation processing parameters were optimised. Maximum enzyme production (9.38 U/ml) was recorded after 96 h of incubation at 35°C, initial pH 5, in submerged culture (200 rpm) utilising 2% (w/v) tannic acid as a sole carbon source. The tannase produced was purified to electrophoretic homogeneity through two-step column chromatography and the purified form remained stable in a pH range of 6–8. Its midpoint of thermal inactivation (T _m) was recorded at 70°C after 60 min of exposure. Maximum tannase activity was enabled at pH 6 and 40°C. Ca²⁺, K⁺, Mg²⁺ and Mn⁺ showed a stimulatory effect while Ba²⁺, Co²⁺, Cu²⁺, Fe³⁺ and Zn⁺ showed a competitive inhibitory effect on enzyme activity. Values of K _m, V _max, K _cat and the molecular mass of the purified enzyme were 0.116 μM ml⁻¹ min⁻¹, 3.57 mM, 1.16 μM ml⁻¹ min⁻¹ and 150 kDa, respectively. The participation of the SH group and carbohydrates in the enzyme structure was also suggested by the results. The stability of the purified and partially purified enzyme at −15°C extended to 13 months.     

Feedback modeling of non-esterified fatty acids in rats after nicotinic acid infusions

A feedback model was developed to describe the tolerance and oscillatory rebound seen in non-esterified fatty acid (NEFA) plasma concentrations following intravenous infusions of nicotinic acid (NiAc) to male Sprague-Dawley rats. NiAc was administered as an intravenous infusion over 30 min (0, 1, 5 or 20 μmol kg⁻¹ of body weight) or over 300 min (0, 5, 10 or 51 μmol kg⁻¹ of body weight), to healthy rats (n = 63), and serial arterial blood samples were taken for measurement of NiAc and NEFA plasma concentrations. Data were analyzed using nonlinear mixed effects modeling (NONMEM). The disposition of NiAc was described by a two-compartment model with endogenous turnover rate and two parallel capacity-limited elimination processes. The plasma concentration of NiAc was driving NEFA (R) turnover via an inhibitory drug-mechanism function acting on the formation of NEFA. The NEFA turnover was described by a feedback model with a moderator distributed over a series of transit compartments, where the first compartment (M ₁) inhibited the formation of R and the last compartment (M _N ) stimulated the loss of R. All processes regulating plasma NEFA concentrations were assumed to be captured by the moderator function. The potency, IC ₅₀, of NiAc was 45 nmol L⁻¹, the fractional turnover rate k _out was 0.41 L mmol⁻¹ min⁻¹ and the turnover rate of moderator k _tol was 0.027 min⁻¹. A lower physiological limit of NEFA was modeled as a NiAc-independent release (k _cap ) of NEFA into plasma and was estimated to 0.032 mmol L⁻¹ min⁻¹. This model can be used to provide information about factors that determine the time-course of NEFA response following different modes, rates and routes of administration of NiAc. The proposed model may also serve as a preclinical tool for analyzing and simulating drug-induced changes in plasma NEFA concentrations after treatment with NiAc or NiAc analogues.     

Physiological response of Cu and Cu mine tailing remediation of <Emphasis Type="Italic">Paulownia fortunei</Emphasis> (Seem) Hemsl

The physiological responses and Cu accumulation of Paulownia fortunei (Seem) Hemsl. were studied under 15.7–157 μmol L⁻¹ Cu treatments in liquid culture for 14 days; the impacts of Cu concentration in the seedlings were evaluated under Cu mine tailing culture with acetic acid and EDTA treatment for 60 days. Results showed that the concentrations of Chl-a, Chl-b and Carotenoids significantly increased (p < 0.05) at 15.7–78.7 μmol L⁻¹Cu treatment and significantly decreased at 157 μmol L⁻¹ treatment after 14 days of Cu exposure. The activities of superoxide dismutase (SOD) and catalase (CAT) significantly increased as Cu levels were enhanced and the activities of both SOD and CAT under 157 μmol L⁻¹ Cu stress were 2.9 and 1.9 times higher than that of control, respectively. The concentrations of proline and soluble sugars in the leaves of P. fortunei significantly increased as the Cu concentrations were elevated. Cu concentrations in roots, stems and leaves of P. fortunei increased significantly as Cu levels increased and reached 1911, 101 and 93 μg g⁻¹ dry weights (DW) at 157 μmol L⁻¹ Cu treatment, respectively. The seedlings of P. fortunei cultivated in Cu tailing experienced unsuccessful growth and loss of leaves in all treatments due to poor nutrition of the Cu tailing. The dry weight of P. fortunei increased under all the treatments of acetic acid after 60 days exposure. However, dry weight significantly decreased under both levels of EDTA. The Cu concentrations increased significantly in roots and decreased in leaves when each was treated with both concentrations of acetic acid. The Cu concentrations in the roots, stems and leaves increased significantly, and the concentrations of Cu in the stems and leaves under the treatment of 2 μmol L⁻¹ EDTA reached 189.5 and 763.1 μg g⁻¹ DW, respectively. The result indicated that SOD, CAT, proline and soluble sugars played an important role in coping with the oxidative stress of copper. Acetic acid could promote growth and EDTA at the experimental levels, which could also enhance Cu absorption and translocation into the stems and leaves of P. fortune. Furthermore, acetic acid and EDTA could be rationally utilized in Cu-contaminated soil.     

Effects of an anionic surfactant (FFD-6) on the energy and information flow between a primary producer (<Emphasis Type="Italic">Scenedesmus obliquus</Emphasis>) and a consumer (<Emphasis Type="Italic">Daphnia magna</Emphasis>)

The effects of a commercially available anionic surfactant solution (FFD-6) on growth and morphology of a common green alga (Scenedesmus obliquus) and on survival and clearance rates of the water flea Daphnia magna were studied. The surfactant-solution elicited a morphological response (formation of colonies) in Scenedesmus at concentrations of 10–100 μl l⁻¹ that were far below the No Observed Effect Concentration (NOEC) value of 1,000 μl l⁻¹ for growth inhibition. The NOEC-value of FFD-6 for colony-induction was 3 μl l⁻¹. Daphnia survival was strongly affected by FFD-6, yielding LC_50–24h and LC_50–48h of 148 and 26 μl l⁻¹, respectively. In addition, clearance rates of Daphnia feeding on unicellular Scenedesmus were inhibited by FFD-6, yielding a 50% inhibition (EC_50–1.5h) at 5.2 μl l⁻¹ with a NOEC of 0.5 μl l⁻¹. When Daphnia were offered FFD-6-induced food in which eight-celled colonies (43 × 29 μm) were most abundant, clearance rates (~0.14 ml ind.⁻¹ h⁻¹) were only 25% the rates of animals that were offered non-induced unicellular (15 × 5 μm) Scenedesmus (~0.56 ml ind.⁻¹ h⁻¹). As FFD-6 concentrations in the treated food used in the experiments were far below the NOEC for clearance rate inhibition, it is concluded that the feeding rate depression was caused by the altered morphology of the Scenedesmus moving them out of the feeding window of the daphnids. The surfactant evoked a response in Scenedesmus that is similar to the natural chemically induced defensive reaction against grazers and could disrupt the natural information conveyance between these plankton organisms.     

Assessment of the potential toxicity of a linear alkylbenzene sulfonate (LAS) to freshwater animal life by means of cladoceran bioassays

The acute and chronic toxic effects of LAS on the cladocerans Daphnia similis, Ceriodaphnia dubia and Ceriodaphnia silvestrii were tested. Both types of toxicity bioassays and the methods of culture and stock maintenance of the test organisms conformed to the recommendations of ABNT (Brazilian Society of Technical Standards), which closely follow the standard methods of USEPA. The results obtained for EC₅₀ (48 h) were: 14.17 mg L⁻¹ for D. similis, 11.84 mg L⁻¹ for C. dubia and 13.52 mg L⁻¹ for C. silvestrii. In the chronic toxicity tests performed on C. dubia and C. silvestrii, there was a significant decrease in the fecundity of the exposed animals; the value of NOEC for C. dubia and C. silvestrii were 1.00 mg L⁻¹ and 2.50 mg L⁻¹, respectively. Cladoceran bioassays provided evidence that LAS concentration as low as 1.00 mg L⁻¹ can damage invertebrate animal life in freshwaters, concentrations that can be found in many eutrophic rivers and reservoirs.     

Effect of Ions on Agitation- and Temperature-Induced Aggregation Reactions of Antibodies

Purpose  The impact of ions on protein aggregation remains poorly understood. We explored the role of ionic strength and ion identity on the temperature- and agitation-induced aggregation of antibodies. Methods  Stability studies were used to determine the influence of monovalent Hofmeister anions and cations on aggregation propensity of three IgG₂ mAbs. The C_H2 domain melting temperature (T _m1) and reduced valence (z*) of the mAbs were measured. Results  Agitation led to increased solution turbidity, consistent with the formation of insoluble aggregates, while soluble aggregates were formed during high temperature storage. The degree of aggregation increased with anion size (F⁻ < Cl⁻ < Br⁻ < I⁻ < SCN⁻ ~ ClO₄ ⁻) and correlated with a decrease in T _m1 and z*. The aggregation propensity induced by the anions increased with the chaotropic nature of anion. The cation identity (Li⁺, Na⁺, K⁺, Rb⁺, or Cs⁺) had no effect on T _m1, z* or aggregation upon agitation. Conclusions  The results indicate that anion binding mediates aggregation by lowering mAb conformational stability and reduced valence. Our observations support an agitation-induced particulation model in which anions enhance the partitioning and unfolding of mAbs at the air/water interface. Aggregation predominantly occurs at this interface; refreshing of the surface during agitation releases the insoluble aggregates into bulk solution. R. Matthew Fesinmeyer and Sabine Hogan have contributed equally to this work.     

Binding Study of the Fluorescence Probe l-Anilino-8-naphthalenesulfonate to Human Plasma and Human and Bovine Serum Albumin Using Potentiometric Titration

The binding of l-anilino-8-naphthalenesulfonate (ANS) to bovine serum albumin (BSA), human serum albumin (HSA), and human plasma has been studied by potentiometric titration utilizing a laboratory constructed ion selective electrode (ISE) of ANS. Three classes of ANS binding sites were found on BSA, HSA, and plasma at 25 and 37°C. Computer analysis of the data resulted in estimates for the association constants, number of binding sites (HSA, BSA), and binding capacity of each class. The association constants for the first class of binding sites at 25°C were found to be 7.53 (±0.59) × 10⁵, 2.70 (±0.20) × 10⁵, and 2.64 (±0.26) × 10⁵ M ^–l for BSA, HSA, and plasma, respectively. Lower values for the association constants of all binding classes were estimated at the higher temperature (37°C). The binding capacity for ANS decreased in the order BSA, plasma, HSA.     

Brain Uptake of Nonsteroidal Anti-Inflammatory Drugs: Ibuprofen, Flurbiprofen, and Indomethacin

Purpose To determine the roles of blood–brain barrier (BBB) transport and plasma protein binding in brain uptake of nonsteroidal anti-inflammatory drugs (NSAIDs)—ibuprofen, flurbiprofen, and indomethacin. Methods Brain uptake was measured using in situ rat brain perfusion technique. Results [¹⁴C]Ibuprofen, [³H]flurbiprofen, and [¹⁴C]indomethacin were rapidly taken up into the brain in the absence of plasma protein with BBB permeability–surface area products (PS_u) to free drug of (2.63 ± 0.11) × 10⁻², (1.60 ± 0.08) × 10⁻², and (0.64 ± 0.05) × 10⁻² mL s⁻¹ g⁻¹ (n = 9–11), respectively. BBB [¹⁴C]ibuprofen uptake was inhibited by unlabeled ibuprofen (K_m = 0.85 ± 0.02 mM, V_max = 13.5 ± 0.4 nmol s⁻¹ g⁻¹) and indomethacin, but not by pyruvate, probenecid, digoxin, or valproate. No evidence was found for saturable BBB uptake of [³H]flurbiprofen or [¹⁴C]indomethacin. Initial brain uptake for all three NSAIDs was reduced by the addition of albumin to the perfusion buffer. The magnitude of the brain uptake reduction correlated with the NSAID free fraction in the perfusate. Conclusions Free ibuprofen, flurbiprofen, and indomethacin rapidly cross the BBB, with ibuprofen exhibiting a saturable component of transport. Plasma protein binding limits brain NSAID uptake by reducing the free fraction of NSAID in the circulation.     

Dynamic features of ecophysiological response of freshwater clam to arsenic revealed by BLM-based toxicological model

The objective of this study was to use a quantitative process-based damage assessment model (DAM) associated with biotic ligand model (BLM) to examine the ecophysiological responses of freshwater clam Corbicula fluminea to waterborne arsenic. We carried out a 14-day exposure experiment to obtain bioaccumulation parameters and a 7-day acute toxicity bioassay to obtain survival data. To investigate the survival dynamics, we examined 2 key parameters characterizing bioaccumulation and damage regulation: capacity to eliminate body arsenic burden and reversible ability to recover the damage. Results show that uptake rate constant of 2.075 ± 0.442 (mean ± SE) ml g⁻¹ d⁻¹ during uptake phase and elimination rate constant was estimated to be 0.1995 ± 0.022 d⁻¹. The derived bioconcentration factor of 10.401 ml g⁻¹ suggests that arsenic has a high potential for bioaccumulation in C. fluminea. Our results show that a negative association between bioaccumulation and the fraction of arsenic binding in biotic ligand at 50% mortality, indicating that C. fluminea with higher arsenic binding in gill biotic ligand at 50% mortality level gives a lower capacity to accumulate bioavailable arsenic. We found a linearly positive correlation between elimination rate and recovery rate constants. Yet, a potential tradeoff between ability to eliminate arsenic and ability to recover the damage is not found. We showed that an ecophysiological significance of C. fluminea exposed to arsenic can be revealed by the elimination–recovery regime. This research may also provide mechanistic insights into the development of biomonitoring organism such as C. fluminea mimicking metal bioaccumulation in a real situation.     

Tolerance and accumulation of lead by species of <Emphasis Type="Italic">Iris</Emphasis> L.

Seedling development, accumulation and distribution of lead (Pb) in Iris lactea var. chinensis (Fisch.) Koidz. and I. tectorum Maxim. were studied using plants grown in sand culture and exposed to 0–10 mmol l⁻¹ concentrations of Pb supplied as Pb(NO₃)₂ for 28 days. A significant reduction in dry weight (dw) of shoots and roots of I. lactea var. chinensis was observed at 6 and 10 mmol l⁻¹, respectively, and a significant reduction in dw of shoots and roots of I. tectorum was observed at 6 mmol l⁻¹. Concentration of Pb in the shoots and roots of I. lacteal var. chinensis exposed to 4 mmol l⁻¹ Pb reached 1,109 μg g⁻¹ and 2,408 μg g⁻¹ dw, respectively. The index of tolerance (IT) of I. lactea var. chinensis among 0–8 mmol l⁻¹ Pb treatments were not significantly different, while those of I. tectorum at 6 mmol l⁻¹ Pb were significantly decreased. The results indicated that I. lactea var. chinensis was more tolerant to Pb than I. tectorum. Sub-cellular localization of Pb in root cells was evaluated using transmission electron microscopy (TEM) and Pb deposits were found along the plasma membrane of some root tip cells of I. lactea var. chinensis treated at 10 mmol l⁻¹ Pb. Deposits of Pd were also observed along the surface, in the root tip cell wall and in the cytoplasm of a few malformed cells of I. tectorum exposed at 10 mmol l⁻¹ Pb treatment. One possible mechanism to explain these observations may be that most cells can maintain normal activities in the plant by sacrificing a small number of cells that accumulate a large amount Pb and show toxicity. Future studies should be designed to test this hypothesis.     

Studies on interaction between Vitamin B12 and human serum albumin

The binding reaction between Vitamin B12 (B12, cyanocobalamin) and human serum albumin (HSA) was investigated by fluorescence quenching, UV–vis absorption and circular dichroism (CD) spectroscopy. Under simulative physiological conditions, fluorescence quenching data revealed that the quenching constants (K_sv) are 3.99 × 10⁴, 4.33 × 10⁴, 4.76 × 10⁴ and 5.16 × 10⁴ M⁻¹ at 292, 298, 304 and 310 K, respectively. The number of binding sites, n is almost constant around 1.0. On the basis of the results of fluorescence quenching the mechanism of the interaction of B12 with HSA has been found to be a dynamic quenching procedure. Thermodynamic parameters ΔH^Θ = −13.38 kJ mol⁻¹, ΔS^Θ = 66.73 J mol⁻¹ K⁻¹ were calculated based on the binding constant. These suggested that the binding reaction was enthalpy and entropy driven, and the electrostatic interaction played major role in stabilizing the reversible complex. The binding distance r = 5.5 nm between HSA and B12 was obtained according to Förster theory of energy transfer. The effect of B12 on the conformation of HSA was analyzed by synchronous fluorescence and CD spectroscopy. Synchronous spectra indicated that the polarity around the tryptophan (Trp214) residues of HSA was decreased and its hydrophobicity was increased; however, the α-helix content of the protein was predominant in the secondary structure but the CD spectra indicated that B12 induced minor conformational changes of HSA.     

Mercury in breeding saltmarsh sparrows (Ammodramus caudacutus caudacutus)

Environmental mercury exposure of birds through atmospheric deposition and watershed point-source contamination is an issue of increasing concern globally. The saltmarsh sparrow (Ammodramus caudacutus) is of high conservation concern throughout its range and the potential threat of mercury exposure adds to other anthropogenic stressors, including sea level rise. To assess methylmercury exposure we sampled blood of the northern nominal subspecies of saltmarsh sparrows (A. c. caudacutus) nesting in 21 tidal marshes throughout most of the species’ breeding range. Blood of tree swallows (Tachycineta bicolor) was sampled concurrently at three of these sites to provide a comparison with a well-studied songbird that is a model species in ecotoxicology. Arithmetic means (±1 SD) ranged from 0.24 ± 0.06 μg g⁻¹ wet weight (ww) in Connecticut to 1.80 ± 0.14 μg g⁻¹ ww in Massachusetts, differing significantly among sites. Comparison to tree swallows indicates that mercury exposure is significantly higher in saltmarsh sparrows, making them a more appropriate bioindicator for assessing risk to methylmercury toxicity in tidal marsh ecosystems.     

Stopped-flow studies on drug-protein binding

Summary We have studied the binding of warfarin to human serum albumin (HSA) with the stopped-flow method. At 37°C the rate constant for the velocity of dissociation of the stable warfarin-HSA complex is 10s^–1 (t _50%=0.07 s). Concentration and temperature dependent association constants for warfarin binding to HSA have been measured (2.5·10⁵ M^–1 s^–1 at 6°C, 9.8·10⁵ M^–1 s^–1 at 22°C and 15.3·10⁵ M^–1 s^–1 at 37°C). Our experimentally obtained relaxation constants are best explained by the existence of 5 equivalent low affinity binding sites for warfarin on the HSA molecule, each capable of conversion into a high affinity site. The measured energy of activation for this conversion is 57.5 kJ M^–1.     

Genotoxicity of crude extracts of cyanobacteria from Taihu Lake on carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>)

Genotoxicity of crude cyanobacteria extracts (CBE) from blooms in Taihu Lake, China toward common carp (Cyprinus carpio) was measured. The primary extracellular product was determined by HPLC to be Microcystin-LR (MC-LR, L for leucine and R for arginine) with an average concentration of 2.4 × 10² μg MC g⁻¹ dry weight of cyanobacteria. Acute toxicity to carp, expressed as the 72-h LC_50, was 53 mg, dw cyanobacteria L⁻¹. Genotoxicity, as determined by the micronucleus (MN) and comet assays were both dose- and time-depended. Deformities of cellular organelles in liver and gill were observed by use of transmission electron microscopy (TEM). The results showed that MC-LR from cyanobacteria from Taihu Lake could induce genotoxic response and tissue-level morphological changes in common carp.     

Studies on reaction of amino acids and triplet thioxanthone derivatives by laser flash photolysis

Summary Excitation of the thioxanthone derivatives (TXs), 2, 4-diethylthioxanthone (DETX) and 2-(2,3-epoxypropyloxy) thioxanthone (ETX) in acetonitrile/water mixture solution (1:1, v/v) upon 355 nm laser flash produced the triplet of TXs (³TXs^*). Characteristic absorption spectra of ³TXs^*(590 nm) were recorded and rate constants of ³TXs^* quenched by O₂ and by its ground state were determined (9.8 × 10⁹ M⁻¹ s⁻¹, 7.3 × 10⁹ M⁻¹ s⁻¹ and 2.6 × 10⁸ M⁻¹ s⁻¹, 2.2 × 10⁸ M⁻¹ s⁻¹ respectively). The reactions of some amino acids oxidized by ³TXs^* were carried out. It has been found that tryptophan (Trp) and tyrosine (Tyr) can quench ³TXs^* via electron transfer process and related quenching rate constants were obtained. ³TXs^* induced protein damage was investigated using electrophoresis and significant levels of dimerisation were observed under aerobic and anaerobic conditions. The influence of photo-sensitizer's structure on photo-oxidation of amino acid and protein has been discussed.     

Toxicity of lead, cadmium and mercury on embryogenesis, survival, growth and metamorphosis of Meretrix meretrix larvae

In order to assess the toxicity of heavy metals on the early development of Meretrix meretrix, the effects of mercury (Hg), cadmium (Cd) and lead (Pb) on embryogenesis, survival, growth and metamorphosis of larvae were investigated. The EC₅₀ for embryogenesis was 5.4 μg l⁻¹ for Hg, 1014 μg l⁻¹ for Cd and 297 μg l⁻¹ for Pb, respectively. The 96 h LC₅₀ for D-shaped larvae was 14.0 μg l⁻¹ for Hg, 68 μg l⁻¹ for Cd and 353 μg l⁻¹ for Pb, respectively. Growth was significantly retarded at 18.5 μg l⁻¹ (0.1 μM) for Hg, 104 μg l⁻¹ (1 μM) for Cd and 197 μg l⁻¹ (1 μM) for Pb, respectively. The EC₅₀ for metamorphosis, similar to 48 h LC₅₀, was higher than 96 h LC₅₀. Our results indicate that the early development of M. meretrix is highly sensitive to heavy metals and can be used as a test organism for ecotoxicology bioassays in temperate and subtropical regions.     

Interaction of 3′ -O-caffeoyl d-quinic acid with human serum albumin

The interaction of chlorogenic acid (CGA) with human serum albumin (HSA) was studied from the viewpoint of thermodynamics and mechanism of binding at pH 6.0. The association constants (K_a) for the HSA-CGA interaction at 10, 25 and 40° C were 6.0 × 10⁴, 9.0 × 10³ and 2 × 10⁴ M^?1, resulting in AG of -6.21, -5.80, -6.32 kcal/mol, respectively. These high K_a -values showed that the interaction between CGA and HSA is strong, endothermic and entropically driven. Binding of chlorogenic acid induces conformational change in HSA as indicated by quenching of fluorescence emission intensity along with a red shift in the emission maxima from 338 to 350 mm. This suggested the involvement of the lone tryptophan residue in the region of binding. Far-ultraviolet circular dichroic data showed a decrease in the α-helical content of HSA from 56 to 50% upon binding of CGA. These data are also supported by the decrease in the apparent Tm of HSA by 4°C upon binding of CGA causing destabilization of the HSA molecule. The kinetics of the interaction involves a single step in the binding, and the kinetic curve attains equilibrium within 180 ± 5 s. Data on caffeic acid (CA) and quinic acid (QA), which are the hydrolysis products of the bidentate CGA molecule, indicate that CA interacts more strongly than CGA. CA binds with an association constant of 8 × 10⁴ M^?1and with a maximum number of binding sites of four. Microcalorimetric investigation of the interaction of these ligands with HSA suggests that the strength of binding follows the order CA?CGA?>QA with a single class of binding sites. The effect of temperature on the binding of CGA to HSA showed that the interaction is dominated by hydrophobic forces and hydrogen bonding.