Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2002-07-02
2004-11-02
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S009000
Reexamination Certificate
active
06811997
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
A method of detecting a metal in a sample is disclosed. More specifically, a method of detecting chromium in an aqueous sample is disclosed.
2. State of the Art
Metals are a significant component of environmental pollutants and contaminants in soil and water. Metals are present at numerous locations around the world, largely from their use in a variety of commercial processes. One of these metals, chromium, is the second most common inorganic contaminant in hazardous waste sites in the Unites States, behind only nitrate. Chromium contaminants are typically present in hazardous waste sites in a trivalent form, Cr(III), and a hexavalent form, Cr(VI), also referred to herein as Cr
3+
and Cr
6+
, respectively. Cr(VI), the more soluble of the two species, is known to be toxic to humans, animals, and plants. On the other hand, Cr(III) is an essential nutrient and one of the best selling dietary supplements in the United States.
The presence of chromium in environmental samples is typically detected by inductively coupled plasma atomic electron spectrometry (“ICP/AES”), inductively couple plasma mass spectrometry (“ICP/MS”), atomic absorption spectroscopy (“AAS”), or wet chemical methods. These techniques have detection limits ranging from parts per trillion to parts per billion. While these techniques are relatively accurate, they are time intensive and require expensive equipment. The current standard analytical technique for total chromium, which includes Cr(VI) and Cr(III), is inductively coupled argon plasma mass spectrometry (“ICP/MS”), which requires expensive capital investment (approximately $200,000) with additional per sample costs in the range of $30-$100. In addition, the equipment is typically massive, immobile and is not suited for use in field testing.
While current techniques detect total chromium, there is no direct, inexpensive method of distinguishing between the Cr(III) and Cr(VI) valence states of chromium. In addition, no direct method of quantitatively measuring Cr(III) exists. One technique for differentiating between Cr(VI) and Cr(III) requires that the samples first be separated by ion-exchange techniques, followed by analysis with AAS or ICP techniques to individually determine the concentrations of Cr(VI) and Cr(III). A second technique uses a colorimetric assay to determine the Cr(VI) concentration. The total chromium is then determined by either AAS or ICP techniques. Then, the Cr(III) concentration is calculated as the difference between total chromium and Cr(VI). However, this method may be inaccurate as it assumes that no other forms of chromium exist in the sample.
Biochemical means of analyzing soil and water samples, such as enzymatic methods, have recently received increased attention. Enzymatic assays are able to detect metals or pesticide inhibitors of the enzyme with greater speed and economy than traditional analytical techniques due to the lower equipment cost, compact size, portability and rapid test times of the enzymatic biosensors. These assays utilize the inhibitory effects that specific metal ions have on the activity of specific enzymes. For example, the enzymatic activities of urease, glucose oxidase, peroxidase, acetyl- or butyryl-cholinesterase, oxalate oxidase, alkaline phosphatase, xanthine oxidase, isocitric or lactate dehydrogenase, and B-fructofuranosidase are inhibited by mercury (“Hg”), copper (“Cu”), silver (“Ag”), cadmium (“Cd”), lead (“Pb”), cobalt (“Co”), manganese (“Mn”), zinc (“Zn”), bismuth (“Bi”), beryllium (“Be”), nickel (“Ni”), Cr(III), and/or Cr(VI). Tadeusz Krawczynski vel Krawczyk,
Chem. Anal
. (
Warsaw
) 43, 135 (1998).
Some of these metals, such as chromium, cadmium, copper, lead, and zinc, are known to substantially inhibit the enzymatic activity of nitrate reductase (“NR”). Even small amounts of these metals and pesticides are known to inhibit NR. NR is an enzyme that catalyzes the conversion of nitrate (“NO
3
−
”) to nitrite (“NO
2
−
”). NR is produced by a variety of animals, plants, and microorganisms, including fungi. For instance,
Aspergillus niger
produces a nicotinamide adenine dinucleotide (“NADH”) or nicotinamide adenine dinucleotide phosphate (“NADPH”) bispecific form of assimilatory NR (EC 1.6.6.2) that catalyzes the reduction of NO
3
−
to NO
2
−
. NR is a homodimer composed of two identical subunits of approximately 100 kDa, each of which contains three cofactors, flavin adenine dinucleotide (“FAD”), heme-iron (heme-Fe) and Mo-molybdopetrin (Mo-MPT) in a 1:1:1 ratio. All known sequences of NR have been found to contain one conserved cysteine (“Cys”) residue that is located in a cytochrome b fragment of the enzyme. Site-directed mutagenesis of the cytochrome b domain of corn leaf NADH:NR showed that this Cys residue is not essential for NADH binding or NADH:NR activity, but is essential for highly efficient catalytic transfer of electrons from the NAD(P)H to FAD. It has also been determined that there are other key Cys residues present in most NR enzymes that are involved in binding the molybdopetrin as well as joining the enzymes subunits. The presence of multiple Cys in NR lends it to be highly sensitive to inhibition by metals because many of these metals, such as Cu and Pb, have high binding affinities for the thiol groups that are present in the Cys side chains.
In evaluating known or suspected sites of metal contamination or while conducting routine drinking water analyses, hundreds of samples may be taken for analysis, many of which may not be contaminated. Thus, it would be desirable to measure Cr(III) by a less expensive method than those currently available. In addition, it would be desirable to speciate Cr(VI) from Cr(III) using a simple, less expensive method than is offered by the current state of the art.
BRIEF SUMMARY OF THE INVENTION
A method of detecting a metal in a sample is disclosed. The method comprises providing the sample potentially comprising a metal. At least a portion of the sample is added to a reagent solution comprising an enzyme and a substrate, where the enzyme is of a type inhibited by the metal. An enzymatic activity in the sample is measured and compared to an enzymatic activity in a control solution to determine the concentration of the metal present in the sample.
In one embodiment, the metal to be detected is chromium. Chromium is detected using a reagent solution comprising nitrate reductase, NADPH, and nitrate. The nitrate reductase converts the nitrate to nitrite, which may be detected colorimetrically.
A method of detecting a metal in a sample comprising a plurality of metals is also disclosed. The method comprises adding a chelating agent to a portion of the sample. The first chelating agent chelates a portion of the metals without chelating the metal to be detected. Another chelating agent is added to the portion of the sample to chelate another portion of the metals without chelating the metal to be detected. When only the metal to be detected remains unchelated, a reagent solution comprising an enzyme and a substrate is added to the portion of the sample. The metal to be detected inhibits the enzyme and is easily detected. The method also comprises quantifying the concentration of the metal in the sample.
A method of detecting a valence state of a metal in a sample is disclosed. The method comprises providing a sample comprising a metal, wherein the metal is present in a plurality of valence states. At least one chelating agent is added to a portion of the sample to chelate at least one portion of the valence states without chelating a valence state of interest. Additional chelating agents are added to the portion of the sample to chelate additional portions of the valence state without chelating the valence state of interest. When the valence state of interest is the only unchelated valence state remaining in the sample, a reagent solution comprising an enzyme and a substrate is added to the portion of the sample, wherein the enzyme is inhibited by the valen
Aiken Abigail M.
Apel William A.
Petersen James N.
Peyton Brent M.
Bechtel BWXT Idaho LLC
Gitomer Ralph
Trask Britt PC
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