We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyamin... more We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyaminocarboxylate ligands (EDTA, DTPA, TTHA, DOTA) and an orthogonal end-group, either a fluorescein molecule or a bismaleimide linker for antibody attachment. Polymer characterization by a combination of (1)H NMR, UV/vis absorption measurements, and thermal gravimetric analysis (TGA) indicated that each chain of the fluorescein-terminated polymers contained one dye molecule. These polymer samples were loaded with three different types of lanthanide ions as well as palladium and platinum ions. The numbers of metal atoms per chain were determined by a combination of UV/vis and conventional ICP-MS measurements. The experiments with lanthanide ions demonstrated that a net anionic charge on the polymer is important for water solubility. These experiments also showed that at least one type of lanthanide ion (La(3+)) is capable of forming a bimetallic complex with pendant DTPA groups. Conditions were developed for loading these polymers with palladium and platinum ions. While these polymers could be conjugated to antibodies, the presence of Pd or Pt ions in the polymer interfered with the ability of the antibody to recognize its antigen. For example, a goat anti-mouse (secondary) antibody labeled with polymers that contain Pd or Pt no longer recognized a primary antibody in a sandwich assay. In mass cytometry assays, these Pd- or Pt-containing MCPs were very effective in recognizing dead cells and provide a new and robust assay for distinguishing live cells from dead cells.
We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyamin... more We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyaminocarboxylate ligands (EDTA, DTPA, TTHA, DOTA) and an orthogonal end-group, either a fluorescein molecule or a bismaleimide linker for antibody attachment. Polymer characterization by a combination of (1)H NMR, UV/vis absorption measurements, and thermal gravimetric analysis (TGA) indicated that each chain of the fluorescein-terminated polymers contained one dye molecule. These polymer samples were loaded with three different types of lanthanide ions as well as palladium and platinum ions. The numbers of metal atoms per chain were determined by a combination of UV/vis and conventional ICP-MS measurements. The experiments with lanthanide ions demonstrated that a net anionic charge on the polymer is important for water solubility. These experiments also showed that at least one type of lanthanide ion (La(3+)) is capable of forming a bimetallic complex with pendant DTPA groups. Conditions were developed for loading these polymers with palladium and platinum ions. While these polymers could be conjugated to antibodies, the presence of Pd or Pt ions in the polymer interfered with the ability of the antibody to recognize its antigen. For example, a goat anti-mouse (secondary) antibody labeled with polymers that contain Pd or Pt no longer recognized a primary antibody in a sandwich assay. In mass cytometry assays, these Pd- or Pt-containing MCPs were very effective in recognizing dead cells and provide a new and robust assay for distinguishing live cells from dead cells.
The glyoxalase I gene (gloA) from Salmonella typhimurium has been isolated in Escherichia coli on... more The glyoxalase I gene (gloA) from Salmonella typhimurium has been isolated in Escherichia coli on a multi-copy pBR322-derived plasmid, selecting for resistance to 3 mM methylglyoxal on Luria-Bertani agar. The region of the plasmid which confers the methylglyoxal resistance in E. coli was sequenced. The deduced protein sequence was compared to the known sequences of the Homo sapiens and Pseudomonas putida glyoxalase I (GlxI) enzymes, and regions of strong homology were used to probe the National Center for Biotechnology Information protein database. This search identified several previously known glyoxalase I sequences and other open reading frames with unassigned function. The clustal alignments of the sequences are presented, indicating possible Zn2+ ligands and active site regions. In addition, the S. typhimurium sequence aligns with both the N-terminal half and the C-terminal half of the proposed GlxI sequences from Saccharomyces cerevisiae and Schizosaccharomyces pombe, suggesting that the structures of the yeast enzymes are those of fused dimers.
The ubiquitous glyoxalase system converts toxic alpha-keto aldehydes into their corresponding non... more The ubiquitous glyoxalase system converts toxic alpha-keto aldehydes into their corresponding nontoxic 2-hydroxycarboxylic acids, utilizing glutathione (GSH) as a cofactor. The first enzyme in this system, glyoxalase I (GlxI), catalyzes the isomerization of the hemithioacetal formed nonenzymatically between GSH and cytotoxic alpha-keto aldehydes. To study the Escherichia coli GlxI enzyme, the DNA encoding this protein, gloA, was isolated and incorporated into the plasmid pTTQ18. Nucleotide sequencing of the gloA gene predicted a polypeptide of 135 amino acids and Mr of 14 919. The gloA gene has been overexpressed in E. coli and shown to encode for GlxI. An effective two-step purification protocol was developed, yielding 150-200 mg of homogeneous protein per liter of culture. Electrospray mass spectrometry confirmed the monomeric weight of the purified protein, while gel filtration analysis indicated GlxI to be a homodimer of 30 kDa. Zinc, the natural metal ion found in the Homo sapiens and Saccharomyces cerevisiae GlxI, had no effect on the activity of E. coli GlxI. In contrast, the addition of NiCl2 to the growth medium or to purified E. coli apo-GlxI greatly enhanced the enzymatic activity. Inductively coupled plasma and atomic absorption analyses indicated binding of only one nickel ion per dimeric enzyme, suggesting only one functional active site in this homodimeric enzyme. In addition, the apoprotein regained maximal activity with one molar equivalence of nickel chloride, indicative of tight metal binding. The effects of pH on the kinetics of the nickel-activated enzyme were also studied. This is the first example of a non-zinc activated GlxI whose maximal activation is seen with Ni2+.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2001
Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine a... more Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overproduced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate semialdehyde to homoserine (Hse) using either NADH or NADPH with kcat/Km in the order of 10(6-7) M(-1) x s(-1) at pH 7.5. The rate constant of the reverse direction (Hse oxidation) was also significant at pH 9.0 (kcat/Km approximately 10(4-5) M(-1) x s(-1)) but was minimal at pH 7.5. Chemical modification of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activity that could be obviated by the presence of substrates. UV difference spectra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consistent with the modification of two histidines (His) per subunit. Amino acid sequence alignment of HSD illustrated the conservation of two His residues among HSDs. These residues, His79 and His309, were substituted to alanine (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while kcat/Km for HSD H309A decreased by almost two orders of magnitude. The recent determination of the X-ray structure of HSD revealed that His309 is located at the dimer interface [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. The His309Ala mutant enzyme was found in very high molecular weight complexes rather than the expected dimer by analytical gel filtration chromatography analysis. Thus the invariant His309 plays a structural rather than catalytic role in these enzymes.
We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyamin... more We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyaminocarboxylate ligands (EDTA, DTPA, TTHA, DOTA) and an orthogonal end-group, either a fluorescein molecule or a bismaleimide linker for antibody attachment. Polymer characterization by a combination of (1)H NMR, UV/vis absorption measurements, and thermal gravimetric analysis (TGA) indicated that each chain of the fluorescein-terminated polymers contained one dye molecule. These polymer samples were loaded with three different types of lanthanide ions as well as palladium and platinum ions. The numbers of metal atoms per chain were determined by a combination of UV/vis and conventional ICP-MS measurements. The experiments with lanthanide ions demonstrated that a net anionic charge on the polymer is important for water solubility. These experiments also showed that at least one type of lanthanide ion (La(3+)) is capable of forming a bimetallic complex with pendant DTPA groups. Conditions were developed for loading these polymers with palladium and platinum ions. While these polymers could be conjugated to antibodies, the presence of Pd or Pt ions in the polymer interfered with the ability of the antibody to recognize its antigen. For example, a goat anti-mouse (secondary) antibody labeled with polymers that contain Pd or Pt no longer recognized a primary antibody in a sandwich assay. In mass cytometry assays, these Pd- or Pt-containing MCPs were very effective in recognizing dead cells and provide a new and robust assay for distinguishing live cells from dead cells.
We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyamin... more We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyaminocarboxylate ligands (EDTA, DTPA, TTHA, DOTA) and an orthogonal end-group, either a fluorescein molecule or a bismaleimide linker for antibody attachment. Polymer characterization by a combination of (1)H NMR, UV/vis absorption measurements, and thermal gravimetric analysis (TGA) indicated that each chain of the fluorescein-terminated polymers contained one dye molecule. These polymer samples were loaded with three different types of lanthanide ions as well as palladium and platinum ions. The numbers of metal atoms per chain were determined by a combination of UV/vis and conventional ICP-MS measurements. The experiments with lanthanide ions demonstrated that a net anionic charge on the polymer is important for water solubility. These experiments also showed that at least one type of lanthanide ion (La(3+)) is capable of forming a bimetallic complex with pendant DTPA groups. Conditions were developed for loading these polymers with palladium and platinum ions. While these polymers could be conjugated to antibodies, the presence of Pd or Pt ions in the polymer interfered with the ability of the antibody to recognize its antigen. For example, a goat anti-mouse (secondary) antibody labeled with polymers that contain Pd or Pt no longer recognized a primary antibody in a sandwich assay. In mass cytometry assays, these Pd- or Pt-containing MCPs were very effective in recognizing dead cells and provide a new and robust assay for distinguishing live cells from dead cells.
The glyoxalase I gene (gloA) from Salmonella typhimurium has been isolated in Escherichia coli on... more The glyoxalase I gene (gloA) from Salmonella typhimurium has been isolated in Escherichia coli on a multi-copy pBR322-derived plasmid, selecting for resistance to 3 mM methylglyoxal on Luria-Bertani agar. The region of the plasmid which confers the methylglyoxal resistance in E. coli was sequenced. The deduced protein sequence was compared to the known sequences of the Homo sapiens and Pseudomonas putida glyoxalase I (GlxI) enzymes, and regions of strong homology were used to probe the National Center for Biotechnology Information protein database. This search identified several previously known glyoxalase I sequences and other open reading frames with unassigned function. The clustal alignments of the sequences are presented, indicating possible Zn2+ ligands and active site regions. In addition, the S. typhimurium sequence aligns with both the N-terminal half and the C-terminal half of the proposed GlxI sequences from Saccharomyces cerevisiae and Schizosaccharomyces pombe, suggesting that the structures of the yeast enzymes are those of fused dimers.
The ubiquitous glyoxalase system converts toxic alpha-keto aldehydes into their corresponding non... more The ubiquitous glyoxalase system converts toxic alpha-keto aldehydes into their corresponding nontoxic 2-hydroxycarboxylic acids, utilizing glutathione (GSH) as a cofactor. The first enzyme in this system, glyoxalase I (GlxI), catalyzes the isomerization of the hemithioacetal formed nonenzymatically between GSH and cytotoxic alpha-keto aldehydes. To study the Escherichia coli GlxI enzyme, the DNA encoding this protein, gloA, was isolated and incorporated into the plasmid pTTQ18. Nucleotide sequencing of the gloA gene predicted a polypeptide of 135 amino acids and Mr of 14 919. The gloA gene has been overexpressed in E. coli and shown to encode for GlxI. An effective two-step purification protocol was developed, yielding 150-200 mg of homogeneous protein per liter of culture. Electrospray mass spectrometry confirmed the monomeric weight of the purified protein, while gel filtration analysis indicated GlxI to be a homodimer of 30 kDa. Zinc, the natural metal ion found in the Homo sapiens and Saccharomyces cerevisiae GlxI, had no effect on the activity of E. coli GlxI. In contrast, the addition of NiCl2 to the growth medium or to purified E. coli apo-GlxI greatly enhanced the enzymatic activity. Inductively coupled plasma and atomic absorption analyses indicated binding of only one nickel ion per dimeric enzyme, suggesting only one functional active site in this homodimeric enzyme. In addition, the apoprotein regained maximal activity with one molar equivalence of nickel chloride, indicative of tight metal binding. The effects of pH on the kinetics of the nickel-activated enzyme were also studied. This is the first example of a non-zinc activated GlxI whose maximal activation is seen with Ni2+.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2001
Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine a... more Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overproduced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate semialdehyde to homoserine (Hse) using either NADH or NADPH with kcat/Km in the order of 10(6-7) M(-1) x s(-1) at pH 7.5. The rate constant of the reverse direction (Hse oxidation) was also significant at pH 9.0 (kcat/Km approximately 10(4-5) M(-1) x s(-1)) but was minimal at pH 7.5. Chemical modification of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activity that could be obviated by the presence of substrates. UV difference spectra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consistent with the modification of two histidines (His) per subunit. Amino acid sequence alignment of HSD illustrated the conservation of two His residues among HSDs. These residues, His79 and His309, were substituted to alanine (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while kcat/Km for HSD H309A decreased by almost two orders of magnitude. The recent determination of the X-ray structure of HSD revealed that His309 is located at the dimer interface [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. The His309Ala mutant enzyme was found in very high molecular weight complexes rather than the expected dimer by analytical gel filtration chromatography analysis. Thus the invariant His309 plays a structural rather than catalytic role in these enzymes.
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Papers by Robert Kinach