Proteinase inhibitory assays of serum using high-performance size exclusion chromatography

A size exclusion column (Spherogel TSK-2000 SW) was utilized in a high-performance size exclusion chromatographic assay to determine the proteinase inhibitory capacity of human sera. Values from assays using this technique agreed well with the standard spectrophotometric inhibitory assays. Nanogram to milligram amounts of protein, namely, alpha 1-proteinase inhibitor, elastase, trypsin, chymotrypsin and their corresponding complexes with the inhibitor, were fractionated in less than 15 min. The nitrated or oxidized alpha 1-proteinase inhibitor was shown to retain its ability to form stable complexes with trypsin or chymotrypsin; however, they lost the inhibitory activity against elastase and instead they behaved as common protein substrates for this enzyme. The present chromatographic procedure was unable to detect any peptide released when the native inhibitor and any of the proteinases reacted to form a complex. Moreover, dissociation of the alpha 1-proteinase inhibitor--elastase complex in an alkaline pH did not result in the formation or release of any peptide.     

Electron-nuclear double resonance spectroscopic evidence that S-adenosylmethionine binds in contact with the catalytically active [4Fe-4S](+) cluster of pyruvate formate-lyase activating enzyme

Pyruvate formate-lyase activating enzyme (PFL-AE) is a representative member of an emerging family of enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (AdoMet) to initiate radical catalysis. Although these enzymes have diverse functions, evidence is emerging that they operate by a common mechanism in which a [4Fe-4S](+) interacts with AdoMet to generate a 5'-deoxyadenosyl radical intermediate. To date, however, it has been unclear whether the iron-sulfur cluster is a simple electron-transfer center or whether it participates directly in the radical generation chemistry. Here we utilize electron paramagnetic resonance (EPR) and pulsed 35 GHz electron-nuclear double resonance (ENDOR) spectroscopy to address this question. EPR spectroscopy reveals a dramatic effect of AdoMet on the EPR spectrum of the [4Fe-4S](+) of PFL-AE, changing it from rhombic (g = 2.02, 1.94, 1.88) to nearly axial (g = 2.01, 1.88, 1.87). (2)H and (13)C ENDOR spectroscopy was performed on [4Fe-4S](+)-PFL-AE (S = (1)/(2)) in the presence of AdoMet labeled at the methyl position with either (2)H or (13)C (denoted [1+/AdoMet]). The observation of a substantial (2)H coupling of approximately 1 MHz ( approximately 6-7 MHz for (1)H), as well as hyperfine-split signals from the (13)C, manifestly require that AdoMet lie close to the cluster. (2)H and (13)C ENDOR data were also obtained for the interaction of AdoMet with the diamagnetic [4Fe-4S](2+) state of PFL-AE, which is visualized through cryoreduction of the frozen [4Fe-4S](2+)/AdoMet complex to form the reduced state (denoted [2+/AdoMet](red)) trapped in the structure of the oxidized state. (2)H and (13)C ENDOR spectra for [2+/AdoMet](red) are essentially identical to those obtained for the [1+/AdoMet] samples, showing that the cofactor binds in the same geometry to both the 1+ and 2+ states of PFL-AE. Analysis of 2D field-frequency (13)C ENDOR data reveals an isotropic hyperfine contribution, which requires that AdoMet lie in contact with the cluster, weakly interacting with it through an incipient bond/antibond. From the anisotropic hyperfine contributions for the (2)H and (13)C ENDOR, we have estimated the distance from the closest methyl proton of AdoMet to the closest iron of the cluster to be approximately 3.0-3.8 A, while the distance from the methyl carbon to the nearest iron is approximately 4-5 A. We have used this information to construct a model for the interaction of AdoMet with the [4Fe-4S](2+/+) cluster of PFL-AE and have proposed a mechanism for radical generation that is consistent with these results.     

Peptide bis-intercalator binds DNA via threading mode with sequence specific contacts in the major groove

BACKGROUND: We previously described a general class of DNA polyintercalators in which 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) intercalating units are connected via peptide linkers, resulting in the first known tetrakis- and octakis-intercalators. We showed further that changes in the composition of the peptide tether result in novel DNA binding site specificities. We now examine in detail the DNA binding mode and sequence specific recognition of Compound 1, an NDI bis-intercalator containing the peptide linker gly-gly-gly-lys. RESULTS: 1H-NMR structural studies of Compound 1 bound to d(CGGTACCG)(2) confirmed a threading mode of intercalation, with four base pairs between the diimide units. The NMR data, combined with DNAse I footprinting of several analogs, suggest that specificity depends on a combination of steric and electrostatic contacts by the peptide linker in the floor of the major groove. CONCLUSIONS: In view of the modular nature and facile synthesis of our NDI-based polyintercalators, such structural knowledge can be used to improve or alter the specificity of the compounds and design longer polyintercalators that recognize correspondingly longer DNA sequences with alternating access to both DNA grooves.     

Alkoxide Clusters of Molybdenum and Tungsten as Templates for Organometallic Chemistry: Comparison with Carbonyl Clusters of the Later Transition Elements

Alkoxide and carbonyl ligands complement each other because they both behave as “π buffers” to transition metals. Alkoxides, which are π donors, stabilize early transition metals in high oxidation states by donating electrons into vacant d_π orbitals, whereas carbonyls, which are π acceptors, stabilize later transition elements in their lower oxidation states by accepting electrons from filled d_π orbitals. Both ligands readily form bridges that span M? M bonds. In solution fluxional processes that involve bridge–terminal ligand exchange are common to both alkoxide and carbonyl ligands. The fragments [W(OR)₃], [CpW(CO)₂], [Co(CO)₃], and CH are related by the isolobal analogy. Thus the compounds [(RO)₃W ? W(OR)₃], [Cp(CO)₂W?W(CO)₂Cp], hypothetical [(CO)₃Co?Co(CO)₃], and HC?CH are isolobal. Alkoxide and carbonyl cluster compounds often exhibit striking similarities with respect to substrate binding—e.g., [W₃(μ₃-CR)(OR′)₉] versus [Co₃(μ₃-CR)(CO)₉] and [W₄(C)(NMe)(OiPr)₁₂] versus [Fe₄(C)(CO)₁₃]—but differ with respect to M? M bonding. The carbonyl clusters use e_g-type orbitals for M? M bonding whereas the alkoxide clusters employ t_2g-type orbitals. Another point of difference involves electronic saturation. In general, each metal atom in a metal carbonyl cluster has an 18-electron count; thus, activation of the cluster often requires thermal or photochemical CO expulsion or M? M bond homolysis. Alkoxide clusters, on the other hand, behave as electronically unsaturated species because the π electrons are ligand-centered and the LUMO metal-centered. Also, access to the metal centers may be sterically controlled in metal alkoxide clusters by choice of alkoxide groups whereas ancillary ligands such as tertiary phosphanes or cyclopentadienes must be introduced if steric factors are to be modified in carbonyl clusters. A comparison of the reactivity of alkynes and ethylene with dinuclear alkoxide and carbonyl compounds is presented. For the carbonyl compounds CO ligand loss is a prerequisite for substrate uptake and subsequent activation. For [M₂(OR)₆] compounds (M = Mo and W) the nature of substrate uptake and activation is dependent upon the choice of M and R, leading to a more diverse chemistry.     

On eigenvalues of symmetric (+1, −1) matrices

To every symmetric matrixA with entries ±1, we associate a graph G(A), and ask (for two different definitions of distance) for the distance ofG(A) to the nearest complete bipartite graph (cbg). Letλ ₁(A),λ ¹ (A) be respectively the algebraically largest and least eigenvalues ofA. The Frobenius distance (see Section 4) to the nearest cbg is bounded above and below by functions ofn −λ ₁ (A), wheren=ord A. The ordinary distance (see Section 1) to the nearest cbg is shown to be bounded above and below by functions ofλ ¹ (A). A curious corollary is: there exists a functionf (independent ofn, and given by (1.1)), such that |λ _i (A) | ≦f(λ ¹(A), whereλ _i (A) is any eigenvalue ofA other thanλ _i (A). This work was supported (in part) by the U.S. Army under contract #DAHC04-C-0023.     

Determination of14C in spent moderator ion-exchange resin from Bruce Nuclear Generating Station A

Spent ion-exchange resins are produced in the purification of coolant and moderator systems during the normal operation of CANDU (Canada deuterium uranium) nuclear reactors. Carbon-14 is a radionuclide of concern in disposal of ion-exchange resins because of its relatively long half-life, its potential high mobility and its ability to be easily incorporated into organisms. Only limited data are presently available on the¹⁴C concentrations of spent from CANDU reactors. To establish a more comprehensive datahase for this radionuclide, concentrations of¹⁴C were determined for two moderator resins from Bruce Nuclear Generating Station A. Mixed bed resins were separated into anion and cation fractions using a sugar solution, and the¹⁴C concentrations were determined for each fraction. Carbon-14-was located predominantly on the anion beads. Samples of anion resin were found to undergo an 81% loss in the¹⁴C concentration over a period of 160 d following the sugar separation procedure. Some evidence is given to suggest this loss in¹⁴C may result from microbial activity. Concentrations and distributions of other predominant radionuclides, such as⁶⁰Co and¹⁵³Gd, are discussed as well.     

Anodic reductions—V The reduction of diketones by unipositive magnesium anodically generated

A series of diketones of the general formula C₆H₅CO(CH₂nCOC₆H₅ has been prepared and subjected to “anodic reduction” in sodium iodide-pyridine solution between magnesium electrodes. In every case hydrolysis of the anolyte following electrolysis yielded a 1:2-diol as the reduction product as evidenced by titration with standard lead tetraacetate solution. The diketones, 1:3-dibenzoylpropane (n = 3) and 1:4-dibenzoylbutane (n = 4), gave cis-1:2-diphenylcyclopentane-1:2-diol and cis-1:2-diphenylcyclohexane-1:2-diol, respectively. On the assumption that the other diketones also gave cyclic 1:2-diols, there is a striking correlation between the initial mean valence number (V_i) of the magnesium entering solution from the anode and the size of the ring; the lowest V_i values were obtained in those instances where the diketones originally in solution gave the most stable cyclic diols. Interpretations are offered for these results and for corrosion phenomena observed.     

Bicyclofulvene—VI : Regioselektive (2 + 2)-cycloadditionen an methylennorbornadien

Ketene imminium ion (4), diphenyl ketene, tosyl isocyanate and tetracyanoethylene add selectively to the semicyclic double bond of 1. This is interpreted to result from the polarity of the semicyclic double bond in 1.     

Isomerization and oxygen atom transfer reactivity in oxo-Mo complexes of relevance to molybdoenzymes

Both dioxo Mo(VI) and mono-oxo Mo(V) complexes of a sterically restrictive N2O heteroscorpionate ligand are found to exist as cis and trans isomers. The thermodynamically stable isomer differs for the two oxidation states, but in each case, we have isolated the kinetically labile isomer and followed its isomerization to the thermodynamically stable form. The Mo(VI) complex is more stable in the cis geometry and isomerizes more than 6 times faster than the Mo(V) complex, which prefers the trans geometry. In OAT reactions with PPh3, the trans isomer of the dioxo-Mo(VI) reacts approximately 20 times faster than the cis isomer. Thus, there are both oxidation state and donor atom dependent differences in isomeric stability and reactivity that could have significant functional implications for molybdoenzymes such as DMSO reductase.