Application of a label-free, gel-free quantitative proteomics method for ecotoxicological studies of small fish species.

Although two-dimensional electrophoresis (2D-GE) remains the basis for many ecotoxicoproteomic analyses, newer non-gel-based methods are beginning to be applied to overcome throughput and coverage limitations of 2D-GE. The overall objective of our research was to apply a comprehensive, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic approach to identify and quantify differentially expressed hepatic proteins from female fathead minnows exposed to fadrozole, a potent inhibitor of estrogen synthesis. Female fathead minnows were exposed to 0 (control), 0.04, and 1.0 µg of fadrozole/L of water for 4 days, and proteomic analysis was performed. Proteins were extracted and digested, and proteolytic peptides were separated via high-resolution one- or two-dimensional (1-D or 2-D) ultrapressure liquid chromatography (UPLC) and analyzed by tandem mass spectrometry. Mass spectra were searched against the National Center for Biotechnology Information (NCBI) ray-finned fish ( Actinopterygii ) database, resulting in identification of 782 unique proteins by single-dimension UPLC. When multidimensional LC analysis (2-D) was performed, an average increase of 1.9× in the number of identified proteins was observed. Differentially expressed proteins in fadrozole exposures were consistent with changes in liver function, including a decline in concentrations of vitellogenin as well as other proteins associated with endocrine function and cholesterol synthesis. Overall, these results demonstrate that a gel-free, label-free proteomic analysis method can successfully be utilized to determine differentially expressed proteins in small fish species after toxicant exposure.

Sediment core versus grab samples: evaluation of contamination and toxicity at a DDT-contaminated site.

Four sites from a stream system near Huntsville, Alabama, contaminated with DDT and its metabolites, were sampled using a coring device. Grab samples were also collected at these and five other sites. Analytical and toxicological evaluations were made on both sets of samples. Core samples provided vertical delineation of toxicity and contamination in sediments, and documented periods of sedimentation with clean material, which appears to be isolating the contaminated sediments from benthic communities. Grab samples yielded less information about the sites. Relationships between DDT concentration and sediment toxicity to Chironomus tentans were similar regardless of the sampling method. Substantial increases in toxicity occurred in most samples when concentrations exceeded 3000 micrograms of DDT residue/g organic carbon.

Toxicity of sediments and sediment pore waters from the Grand Calumet River-Indiana Harbor, Indiana area of concern.

The assessment of contaminated sediments is a difficult task due to the complex nature of the sediment matrix and the potential for exposure of aquatic organisms to in-place contaminants via several routes. Differential species sensitivity also precludes the completion of a meaningful environmental assessment with only one species. Therefore, a battery of assays approach with the Microtox assay, 48 hr Daphnia magna and Ceriodaphnia dubia tests and a 10-day Chironomous tentans test was used to evaluate the toxicity of sediment pore waters and whole sediments from the Grand Calumet River-Indiana Harbor, Indiana area of concern. All toxicity tests indicated that the test fractions (pore water, whole sediment) from each study site were toxic to the test species. A toxic units (TU) approach was used to compare measured TU from each assay with calculated TU based on chemical analyses of pore waters and whole sediments and the results of reference toxicant tests. Based on the results of these analyses, ammonia, polycyclic aromatic hydrocarbons, metals, petroleum hydrocarbons, and bicarbonate ion appear to be the major contaminants of environmental significance to benthic invertebrates within the study area.

Sediment pore water toxicity identification in the lower Fox River and Green Bay, Wisconsin, using the Microtox assay.

Microtox assays with two different methods of osmotic adjustment were used to assess the toxicity of pore waters from 13 sediment samples collected from the Fox River watershed in Wisconsin. No toxicity was observed in Microtox assays osmotically adjusted with NaCl; however, 15-min EC50 values for assays osmotically adjusted with sucrose ranged from 52 to 63% pore water. Un-ionized ammonia accounted for a large part of the observed toxicity, but, based on a toxic units approach, did not account for all observed toxicity. Metals (Cu, Zn) and an unidentified compound(s) may potentially contribute to the observed effects in Microtox assays osmotically adjusted with sucrose. The use of alternative osmotic adjustment techniques in the Microtox assay is one potentially useful tool for elucidating several classes of compounds responsible for effects observed in toxicity assays.

Synthesis of serine-AMC-carbamate: a fluorogenic tryptophanase substrate.

A new fluorogenic substrate for the pyridoxal 5'-phosphate-dependent enzyme tryptophanase is described. L-Serine, which is linked to 7-amino-4-methylcoumarin through an O-carbamoyl tether, serves as a substrate for the enzyme. The released moiety, 7-amino-4-methylcoumarin (AMC), can be detected by either absorbance (355 nm) or fluorescence (excitation 365 nm/emission 440 nm). Kinetic constants were measured using each of these techniques: Km = 85 +/- 20 microM, Vmax = 2.9 +/- 0.4 mumol/min/mg (fluorescence) and Km = 129 +/- 21 microM, Vmax = 3.1 +/- 0.3 mumol/min/mg (absorbance). The Vmax for serine-AMC-carbamate is approximately 1.9 times faster than that of the natural substrate, tryptophan. Using fluorescence detection, solutions containing 10(-3) units of activity could be routinely assayed.

Dual-enzyme cascade--an amplified method for the detection of alkaline phosphatase.

A method in which a two-enzyme cascade is used for rapid and sensitive detection of alkaline phosphatase is described. A masked inhibitor, 4-(3-oxo-4,4,4-trifluorobutyl)phenyl phosphate, is dephosphorylated by the action of alkaline phosphatase. The resulting compound, 1,1,1-trifluoro-4-(4-hydroxyphenyl)-butan-2-one, acts as a potent inhibitor of the second enzyme, a liver carboxylesterase. A determination of the residual esterase activity provides a highly sensitive indication of the original phosphatase concentration. The sensitivity of this dual-enzyme cascade is approximately 125-fold greater than that observed for the direct detection of phosphatase activity with p-nitrophenyl phosphate.

Studies on Ca(II) binding to gamma-carboxyglutamic acid. Use of thermal decarboxylation to probe metal ion/gamma-carboxyglutamic acid interactions.

The thermal decarboxylation of N-benzyloxycarbonyl-L-gamma-carboxyglutamic acid alpha-methyl ester [Z)-L-Gla-OMe) has been studied. In the presence of increasing amounts of calcium or magnesium ions, lyophilized powders of (Z)-L-Gla-OMe exhibit a corresponding increase in thermal stability. Both magnesium and calcium form relatively tight, thermally stable complexes with (Z)-L-Gla-OMe at high metal ion concentrations. Differences between Ca(II) and Mg(II) binding are noted at low metal ion concentrations, where (Z)-L-Gla-OMe is in excess. Under these conditions, complex formation with Mg(II) apparently favors a 2:1 Gla-magnesium ion complex in which both Gla residues are unstable to thermal decarboxylation. Calcium ion complexes, however, are found to favor a 3:1 Gla-calcium ion complex in which 1 of the 3 Gla residues is thermally stable.

Synthesis of a gamma-carboxyglutamic acid containing heptapeptide corresponding to bovine prothrombin residues 17-23.

The vitamin K-dependent proteins involved in blood coagulation processes each contain a small cystine loop with the sequence -Gla-Cys-X-Gla-Gla-X-Cys-. A method for the synthesis of the heptapeptide derivative representing the 17-23 sequence of bovine prothrombin, Z-Gla-Cys-Leu-Gla-Gla-Pro-Cys-OBzl, has been developed. The 25Mg2+ n.m.r. spectrum of the heptapeptide derivative has been obtained and is compared to the n.m.r. spectra obtained from the interaction of 25Mg2+ with Z-Gla-OMe and Z-Gla-Gla-OMe.

Relative spectral response as a function of sequential ligand binding.

A unique method is presented for the determination of the critical number of ligands that must bind to a macromolecule to elicit a spectroscopic response. This method is based on analysis of ligand binding data. For example, four Ca2+ and two Mg2+ ions are necessary for mirroring the relative decrease in the intrinsic fluorescence of bovine prothrombin fragment 1. For application of the method, ligand loading and relative spectroscopic response data must be measured over a full range of concentrations.

The effect of divalent metal ions on the electrophoretic mobility of bovine prothrombin and bovine prothrombin fragment 1.

Examination of metal ion-dependent effects on the electrophoretic mobility of bovine prothrombin and fragment 1 provides a useful and sensitive method for investigation of conformational processes in these proteins. Utilization of this method reveals a conformational change in bovine prothrombin and fragment 1 which occurs at low metal ion concentrations. Equilibrium dialysis studies indicate that the metal ion-induced shape change occurs concomitant with binding of a single calcium ion/molecule of prothrombin or fragment 1. Mixed metal electrophoretic mobility studies with Mg2+ and Ca2+ have demonstrated the "synergistic" effect for fragment 1 observed by others. Mixed metal equilibrium dialysis has provided experimental support for this observation and allows us to conclude that two tight Ca2+ sites are not affected by low Mg2+ concentrations and that the third Ca2+ site is also a tight site for Mg2+. Thus, at low Mg2+ concentrations and upon the addition of Ca2+, there are effectively three tight sites; consequently more Ca2+ will bind to the protein at lower total Ca2+ ion concentrations.

A method for specific chemical modification of gamma-carboxyglutamic acid residues in proteins.

A method for the chemical modification of gamma-carboxyglutamic acid (Gla) residues in proteins is introduced that has the combined advantages of mildness, a high degree of specificity, and the ability to introduce a radiolabel at modification sites for ease in quantitation. Unlike other Gla modification procedures which are performed in the lyophilized state at 110 degrees C, this procedure is carried out in solution at 37 degrees C. The addition of morpholine and formaldehyde to a slightly acidic solution of bovine prothrombin fragment 1 (residues 1-156) results in the conversion of Gla residues to gamma- methyleneglutamic acid (gamma- MGlu ). The extent of modification is controlled by the relative amounts of modification reagents to protein. A 100-fold molar excess of reagents to fragment 1 produced a protein molecule containing two gamma- MGlu residues, while a modification run at 10,000-fold molar excess of reagents to protein yielded fragment 1 containing eight gamma- MGlu residues per molecule. The specificity of this modification is illustrated by the interaction of native and modified protein with antibody populations directed against fragment 1. Native fragment 1, 8 gamma- MGlu fragment 1, and 2 gamma- MGlu fragment 1 show fairly similar behavior toward whole anti-fragment 1 serum. Differential behavior was exhibited by the native and modified proteins toward a subpopulation of antibodies specific to the calcium ion conformation of fragment 1. Unmodified fragment 1 displayed a strong affinity for these antibodies; however, the 2 gamma- MGlu fragment 1 exhibited a moderate affinity and the 8 gamma- MGlu fragment 1 did not bind to these antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)