Type I IFNs are unusually pleiotropic cytokines that bind to a single heterodimeric receptor and have potent antiviral, antiproliferative, and immune modulatory activities. The diverse effects of the type I IFNs are of differential therapeutic importance; in cancer therapy, an enhanced antiproliferative effect may be beneficial, whereas in the therapy of viral infections (such as hepatitis B and hepatitis C), the antiproliferative effects lead to dose limiting bone marrow suppression. Studies have shown that various members of the natural IFN-alpha family and engineered variants, such as IFN-con1, vary in the ratios between various IFN-mediated cellular activities. We used DNA shuffling to explore and confirm the hypothesis that one could simultaneously increase the antiviral and Th1-inducing activity and decrease the antiproliferative activity. We report IFN-alpha hybrids wherein the ratio of antiviral:antiproliferative and Th1-inducing: antiproliferative potencies are markedly increased with respsect to IFN-con1 (75- and 80-fold, respectively). A four-residue motif that overlaps with the IFNAR1 binding site and is derived by cross breeding with a pseudogene contributes significantly to this phenotype. These IFN-alphas have an activity profile that may result in an improved therapeutic index and, consequently, better clinical efficacy for the treatment of chronic viral diseases such as hepatitis B virus, human papilloma virus, HIV, or chronic hepatitis C.

N-Acetylation is a modification of glyphosate that could potentially be used in transgenic crops, given a suitable acetyltransferase. Weak enzymatic activity (k(cat) = 5 min(-1), K(M) = 1 mM) for N-acetylation of glyphosate was discovered in several strains of Bacillus licheniformis (Weigmann) Chester by screening a microbial collection with a mass spectrometric assay. The parental enzyme conferred no tolerance to glyphosate in any host when expressed as a transgene. Eleven iterations of DNA shuffling resulted in a 7000-fold improvement in catalytic efficiency (k(cat)/K(M)), sufficient for conferring robust tolerance to field rates of glyphosate in transgenic tobacco and maize. In terms of k(cat)/K(M), the native enzyme exhibited weak activity (4-450% of that with glyphosate) with seven of the common amino acids. Evolution of the enzyme towards an improved k(cat)/K(M) for glyphosate resulted in increased activity toward aspartate (40-fold improved k(cat)), but activity with serine and phosphoserine almost completely vanished. No activity was observed among a broad sampling of nucleotides and antibiotics. Improved catalysis with glyphosate coincided with increased thermal stability.

The herbicide glyphosate is effectively detoxified by N-acetylation. We screened a collection of microbial isolates and discovered enzymes exhibiting glyphosate N-acetyltransferase (GAT) activity. Kinetic properties of the discovered enzymes were insufficient to confer glyphosate tolerance to transgenic organisms. Eleven iterations of DNA shuffling improved enzyme efficiency by nearly four orders of magnitude from 0.87 mM(-1) min(-1) to 8320 mM(-1) min(-1). From the fifth iteration and beyond, GAT enzymes conferred increasing glyphosate tolerance to Escherichia coli, Arabidopsis, tobacco, and maize. Glyphosate acetylation provides an alternative strategy for supporting glyphosate use on crops.

Hydroxyl radical-effected protium/deuterium ((1)H/(2)H) exchange into the C-H bonds present in peptides has been used to identify the site of hydrogen atom abstraction by hydroxyl radical. Radiolysis of anaerobic, N(2)O-saturated D(2)O solutions containing peptide and dithiothreitol generates a hydroxyl radical that mediates (1)H/(2)H exchange into the side chains of peptides of up to 66 atom % excess (2)H. The (1)H/(2)H exchange is determined by measuring the isotope ratio, [M + H + 1](+)/[M + H](+), of the peptide using electrospray ionization-mass spectrometry. The (1)H/(2)H exchange within each residue of the peptide was determined by measuring the isotope ratio of each isolated dansyl amino acid following hydrolysis and derivatization. Generation of 0.40 mM hydroxyl radical effected (1)H/(2)H exchange into each of the five different residues of (Ala(2))-leucine enkephalin (YAGFL). The propensity of the residues to undergo exchange was L > Y > A congruent with F > G, independent of whether they were radiolyzed separately or as the peptide. The minimal exchange into glycine suggests that reaction of hydroxyl radical with the side chain hydrogens predominates over reaction with the polypeptide alpha-hydrogens. The ability of radiolysis to effect (1)H/(2)H exchange into a larger peptide, SNEQKACKVLGI, was also demonstrated.

A general method of unequivocally identifying and obtaining sequence information on cross-linked peptides derived by proteolytic digestion of cross-linked proteins has been developed. The method is based on isotopic labeling of α-amino groups with 2,4-dinitrofluorobenzene (DNFB) coupled with electrospray ionization mass spectrometry. Proteins containing covalent cross-link(s) are reductively methylated to convert lysine residues to dimethyl lysine. The methylated protein is partially hydrolyzed and the liberated α-amino termini are derivatized with an equimolar mixture of DNFB and [2H3]DNFB. Dinitrophenyl (DNP)-labeled peptides may be fractionated into mono- and bis-DNP pools by chromatography on phenyl media. The bis-DNP peptides are further separated by reverse-phase HPLC and analyzed by electrospray ionization mass spectrometry. The molecular ions of cross-linked peptides are unambiguously identified as 1:2:1 triplets in the mass spectrum resulting from the binomial distribution of isotopic label in the bis-DNP derivative. Sequence information can be elucidated from the unique product ion patterns which are generated from in-source fragmentation at an elevated cone voltage. Analysis of the disulfide cross-linked peptide (VTÇG)2 was undertaken as a proof of concept and the generality of the method was demonstrated by isolating and sequencing the isopeptide bond of polyubiquitin.

Iron granules (< 10 μ) (Fe°‐H 2 O) and palladium‐treated iron granules (Pd/Fe°‐H 2 O) in contact with water have been tested as a potential means to dechlorinate chloromethanes (CCl 4 , CHCl 3 , and CH 2 Cl 2 ) that are commonly generated in teaching chemistry laboratories. Palladium treatment enhanced the rate of dechlorination of CCl 4 by a factor of about seven compared to the untreated Fe°‐H 2 O, but the potential toxic effect of palladium remains a concern. Oxygen in the aqueous solution reduced the dechlorination rates of CCl 4 with both Fe°‐H 2 O and Pd/Fe°‐H 2 O by at least a factor of three. Nevertheless, from the consideration of remediating solvent wastes, both systems appear suitable for treating CCl 4 and possibly CHCl 3 wastes, even in the presence of oxygen. The reactivities of the three chloromethanes toward the Fe°‐H 2 O were vastly different. The dechlorination rate of CCl 4 was by far the highest, followed by that of CHCl 3 , and CH 2 Cl 2 was virtually unreactive toward the Fe°‐H 2 O system. Dechlorination reactions of CCl 4 and CHCl 3 were systematically examined on both Fe°‐H 2 O and Pd/Fe°‐H 2 O. On the basis of the data, it appears that CCl 4 is successively dechlorinated to form CH 4 via the formation of partially dechlorinated intermediates. Proton transfer to various intermediates produces the chloromethanes: CHCl 3 , CH 2 Cl 2 , and CH 3 Cl. Furthermore, CH 4 formation, independent of the proton‐transferring reactions, appears to be selectively stimulated by palladium (Pd/Fe°‐H 2 O).

An on-line immunoaffinity extraction with liquid chromatography/membrane introduction mass spectrometry (IAE/LC/MIMS) method for the determination of BTEX compounds in complex sample matrixes is described. This method uses an immunoaffinity column (1 mm i.d. x 20 mm) for on-line sample cleanup and enrichment, a 5-micron C18 trapping column (2 mm i.d. x 20 mm) for analyte focusing, a 3-micron C18 analytical column (3.2 mm i.d. x 100 mm) for separation, and a membrane introduction mass spectrometer for quantitation. The immunoaffinity column was evaluated in terms of binding capacity, recovery, and enrichment factor. The method was optimized for the determination of BTEX compounds in a mixture of 30 volatile organic compounds, which showed no matrix interference and a dramatic improvement of the detection limit over that of the LC/MIMS method (up to 474-fold). This method was also used for the determination of BTEX compounds in several gasoline-contaminated water samples, and the results were compared with the EPA reference methods.

Many Ti(IV) metallocenes Cp2TiX2 and CpTiX3 (X = halogen, Cp = eta5-C5H5) exhibit an intense, long-lived charge transfer phosphorescence at 77 K arising from the radiative decay of a Cp --> Ti charge transfer triplet excited state. The phosphorescence band can be shifted systematically by varying X or by replacing Cp with Cp* = eta5-C5(CH3)5. Trends in the phosphorescence spectra follow closely related trends previously noted in the electronic absorption spectra, photoelectron spectra, and photochemical behavior of these metallocenes. The conspicuous absence of phosphorescence emission in Cp2TiI2 and CpTi(CH3)2 correlates with the photochemistry and spectroscopy of these systems; the Cp --> Ti excitation is no longer the lowest energy excitation. Low temperature electronic absorption spectroscopy is utilized to probe the I --> Ti and Cp --> Ti charge transfer excitations of Cp2TiI2. It is proposed that the presence or absence of charge transfer phosphorescence can be used as a diagnostic to determine the relative energy ordering of the valence Cp and X orbitals in Ti(IV) metallocenes.