Isotherms for adsorption of cellobiohydrolase I and II fromtrichoderma reesei on microcrystalline cellulose

Adsorption to microcrystalline cellulose (Avicel) of pure cellobiohydrolase I and II (CBH I and CBH II) fromTrichoderma reesei has been studied. Adsorption isotherms of the enzymes were measured at 4‡C using CBH I and CBH II alone and in reconstituted equimolar mixtures. Several models (Langmuir, Freundlich, Temkin, Jovanovic) were tested to describe the experimental adsorption isotherms. The isotherms did not follow the basic (one site) Langmuir equation that has often been used to describe adsorption isotherms of cellulases; correlation coefficients (R²) were only 0.926 and 0.947, for CBH I and II, respectively. The experimental isotherms were best described by a model of Langmuir type with two adsorption sites and by a combined Langmuir-Freundlich model (analogous to the Hill equation); using these models the correlation coefficients were in most cases higher than 0.995. Apparent binding parameters derived from the two sites Langmuir model indicated stronger binding of CBH II compared to CBH I; the distribution coefficients were 20.7 and 3.7 L/g for the two enzymes, respectively. The binding capacity, on the other hand, was higher for CBH I, 1.0 Μmol (67 mg) per gram Avicel, compared to 0.57 Μmol/g (30 mg/g) for CBH II. The isotherms when analyzed with the combined Langmuir-Freundlich model indicated presence of unequal binding sites on cellulose and/or negative cooperativity in the binding of the enzyme molecules.     

Tailoring pore properties of MCM-48 silica for selective adsorption of CO2

Four different types of amine-attached MCM-48 silicas were prepared and investigated for CO(2) separation from N(2). Monomeric and polymeric hindered and unhindered amines were attached to the pore surface of the MCM-48 silica and characterized with respect to their CO(2) sorption properties. The pore structures and amino group content in these modified silicas were investigated by XRD, FT-IR, TGA, N(2) adsorption/desorption at 77 K and CHN/Si analysis, which confirmed that in all cases the amino groups were attached to the pore surface of MCM-48 at 1.5-5.2 mmol/g. The N(2) adsorption/desorption analysis showed a considerable decrease of the pore volume and surface area for the MCM-48 silica containing a polymeric amine (e.g., polyethyleneimine). The CO(2) adsorption rates and capacities of the amine-attached MCM-48 samples were studied employing a sorption microbalance. The results obtained indicated that in addition to the concentration of surface-attached amino groups, specific interactions between CO(2) and the surface amino groups, and the resultant pore structure after amine group attachment have a significant impact on CO(2) adsorption properties of these promising adsorbent materials.     

Formation and stability of a Janus-Wedge type of DNA triplex

A new type of DNA targeting with the formation of a Janus-Wedge (J-W) triple helix is described. The "wedge" residue (W) attached to a PNA backbone is designed to insert itself into double-stranded DNA and base pair with both Watson-Crick faces. To study the stability of such an assembly, we have examined the formation of the J-W triplex with dC8 - T8 target sequence. The use of this target sequence permits the study of this new helix form without competing Watson-Crick interactions between the two target residues. Studies indicate that the W strand binds to both target strands, with defined polarity and a stability (-15.2 kcal/mol) that is roughly the sum of the two independent duplex interactions.     

ENHANCED SUSCEPTIBILITY TO HPD-SENSITIZED PHOTOTOXICITY AND CORRELATED RESISTANCE TO TRYPSIN DETACHMENT IN SV-40 TRANSFORMED IMR-90 CELLS

Abstract— The response of a normal human cell strain, IMR-90 and a line derived from it by SV40 transformation were compared after treatment in vitro with hematoporphyrin derivative (HPD) and red light. Transformed cells were inactivated at a significantly higher rate than normal cells when assayed by clonogenic survival. Co-treatment with HPD and light also induces cellular resistance to enzymatic cleavage by trypsin; transformed cells exhibit a greater resistance than the normal cells to detachment from the growing surface. These data indicate that transformed cells may possess intrinsic characteristics that render them more sensitive than normal cells to HPD-induced phototoxicity and that the plasma membrane may be the site of differential toxicity.     

Geometry and force constant determination from correlated wave functions for polyatomic molecules: Ground states of H2O and CH2

Ab initio calculations including electron correlation are reported for the water and methylene molecules as a function of geometry. A large contracted gaussian basis set is used and the multiconfiguration wave functions, optimized by the iterative natural orbital procedure, include 277 and 617 configurations for H₂O and CH₂ respectively. The method of selecting configurations, yielding first-order wave functions, is discussed in some detail. For H₂O, the SCF geometry is r=0,942 Å, =105,8°, the correlated result is r=0,968 Å, =103,2°, and the experimental r=0,957 Å, =104,5°. The water stretching force constants, in millidynes/Å, are 8,72 (SCF), 8,75 (CI), and 8,4 (experiment). Bending force constants are 0,88 (SCF), 0,83 (CI), and 0,76 (experiment). For methylene the SCF geometry is r=1,072 Å, =129,5°, while the result from first-order wave functions is r=1,088 Å, =134°. The predicted CH₂ force constants are 6,16 (SCF) and 6,13 (CI) for stretching and 0,44 (SCF) and 0,33 (CI) for bending.

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Zusammenfassung Es wird über ab intito-Rechnungen mit Berücksichtigung der Elektronenkorrelation berichtet, die an Wasser- und Methylenmolekülen als Funktion der Geometrie durchgeführt worden sind. Dazu benutzt man einen großen kontrahierten Gauß-Basissatz. Die Multikonfigurationswellenfunktionen, die unter Benutzung von natürlichen Orbitalen nach der iterativen Prozedur optimiert werden, enthalten für H₂O 277 Konfigurationen und für CH₂ 617. Die Auswahlmethode, die zu Wellenfunktionen 1. Ordnung führt, wird diskutiert. Im Falle des Wassers erhält man die SCF-Geometrie zu r=0,942 Å, =105,8°, das korrelierte Resultat ist: r=0,968 Å, =103,2° und das experimentelle r=0,957 Å, =104,5°. Für Wasser ergeben sich die Valenzkraftkonstanten (in Millidyn Å^–1) 8,72 (SCF), 8,75 (CI) und 8,4 (Experiment). Die Deformationskonstanten sind 0,88 (SCF), 0,83 (CI) und 0,76 (Experiment). Im Falle des Methylens ist die SCF-Geometrie r=1,072 Å, =129,5°, während man mit Wellenfunktionen 1. Ordnung r=1,088 Å und =134° erhält. Die CH₂-Kraftkonstanten werden für die Valenzschwingung zu 6,16 (SCF) und 6,13 (CI) bzw. für die Deformationsschwingung zu 0,44 (SCF) und 0,33 (CI) vorausgesagt.

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Work performed under the auspices of the U.S. Atomic Energy Commision.

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Supported by the grants from the Research Corporation and the University of California Committee on Research.     

Broadband rotational resonance in solid state NMR spectroscopy

A new technique for restoring nuclear magnetic dipole-dipole couplings under magic-angle spinning (MAS) in solid state nuclear magnetic resonance (NMR) spectroscopy is described and demonstrated. In this technique, called broadband rotational resonance (BroBaRR), the coupling between a pair of nuclear spins with NMR frequency difference close (but not necessarily equal) to the MAS frequency is restored by the application of a train of weak radio-frequency pulses at a carrier frequency close to the average of the two NMR frequencies. Phase or amplitude modulation of the pulse train at half the MAS frequency splits the carrier into sidebands close to the two NMR frequencies. The pulse train then removes offsets from the exact rotational resonance condition, leading to dipolar recoupling over a bandwidth controlled by the amplitude of the pulse train. (13)C NMR experiments on uniformly (15)N,(13)C-labeled L-valineHClH(2)O powder validate the theoretical analysis. BroBaRR will be useful in studies of molecular structures by solid state NMR, for example in the detection of long-range couplings between carbons in uniformly labeled organic and biological materials.     

Synthesis and characterization of random poly(amide-sulfonamide)s. II. Polymers from unsymmetrical diamine monomers

Novel poly(amide-sulfonamide)s have been prepared by reacting terephthalic, isophthalic, and sebacic acid and their acid chlorides with variously substituted diamines containing preformed sulfonamide linkages utilizing solution polymerization techniques. Inherent viscosities of the prepared polymers varied from 0.22 to 1.21 dL/g. Those having inherent viscosities greater than 0.4 dL/g formed clear, tough, flexible films. Glass transition temperatures ranged from 87 to 273°C. Thermogravimetric analyses of the polymers showed moderate thermal stability.     

Benzannelated analogs of phenanthro[1,2-b]- and [2,1-b]thiophene: Synthesis and structural characterization by two-dimensional NMR and X-ray techniques

Syntheses of benzo[3,4]phenanthro[1,2-b]thiophene, benzo[3,4]phenanthro[2,1-b]thiophene and their 1-methyl analogs are reported as potential constituents of solvent refined coal liquids and for mutagenicity testing. The attempted synthesis of the 13-methyl analogs which gave the 11-methyl isomers is also described. Total assignments of the ¹H- and ¹³C-nmr spectra based on long range optimized heteronuclear protoncarbon two-dimensional chemical shift correlation are reported. Carbon assignments obtained for benzo[3,4]-phenanthro[1,2-b]thiophene using this approach were confirmed with a 125 MHz ¹³C–¹³C INADEQUATE spectrum. X-Ray crystal structures were determined for benzo[3,4]phenanthro[1,2-b]thiophene and 1-methyl-benzo[3,4]phenanthro[2,1-b]thiophene. Both molecules were helically distorted from planarity. Close intramolecular contacts between the bay region H1–H13 and ClMe-H13 of 2.03 and 2.28 Å, respectively, were responsible for the distortions. There were no close intermolecular contacts of <3.5Å. both molecules refined to an R value of <0.05.     

Solid-state NMR spectroscopy method for determination of the backbone torsion angle psi in peptides with isolated uniformly labeled residues

We demonstrate a solid-state nuclear magnetic resonance technique, with the acronym ROCSA-LG, for the determination of backbone torsion angles psi in peptides with multiple, but isolated, uniformly labeled residues. The method correlates the 13C' chemical shift anisotropy and the 13Calpha-1Halpha heteronuclear dipolar tensors within a single uniformly labeled residue in a two-dimensional (2D) experiment. The technique requires the measurement of only five 2D spectra and is compatible with high-speed magic-angle spinning. Experimental results are presented for the 17-residue alpha-helical peptide MB(i+4)EK and for amyloid fibrils formed by the 15-residue peptide Abeta11-25.     

PHOTOBLEACHING OF MONO-l-ASPARTYL CHLORIN e6 (NPe6): A CANDIDATE SENSITIZER FOR THE PHOTODYNAMIC THERAPY OF TUMORS

Abstract— Most sensitizers used for the photodynamic therapy (PDT) of tumors photobleach on illumination. Thus, it is of interest to examine the photobleaching behavior of new sensitizers proposed for use in PDT. This report surveys the quantum yields and kinetics of the photobleaching of mono- l -aspartyl chlorin e₆ (NPe6), a hydrophilic chlorin that has many of the photoproperties desirable in a sensitizer for clinical PDT. It is a very effective sensitizer for the PDT of several types of model tumors in animals and is now in Phase I clinical trials. The quantum yield of NPe6 photobleaching in pH 7.4 phosphate buffer in air was 8.2 × 10⁻⁴; this is greater than the yields for typical porphyrin photosensitizers. For example, the yields for hematoporphyrin and uroporphyrin are 4.7 × 10 ⁵ and 2.8 × 10⁻⁵, respectively. The yield decreased significantly in organic solvents of low dielectric constant. The Sn derivative of NPe6 was more light stable than NPe6 (yield = 5.7 × 10 ⁻⁶), while the Zn derivative was more sensitive (yield = 1.9 × 10⁻²). Oxygen appeared to be necessary for the photobleaching of NPe6; however, bleaching was not inhibited by 100 mM azide, an efficient quencher of singlet oxygen. The photooxidizable substrates cysteine, dithiothreitol and furfuryl alcohol increased the quantum yield of photoblcaching two- to four-fold, while the electron acceptor, met-ronidazole, increased it almost six-fold. Photobleaching yields for several other chlorins were also measured.