Stark modulation of the electronic spectrum of pyridine in benzene at 1.6 K

The Stark-modulated spectra of the OO band of the ¹B₁ ← ¹N₁ (n → π*) transition of pyridine in single crystals of benzene were studied for all principal directions of the applied field. The molecules were found to be oriented with their C₂ axis nearly parallel to the benzene CH bond direction closest to the crystallographic b-axis. The Lorentz field corrected dipole moment change on exciation was found to be 2.6 ± 0.1 debye.     

N-t-butyliodoacetamide and iodoacetanilide: two new cysteine alkylating reagents for relative quantitation of proteins

The synthesis and application of two new alkylating reagents, N-tert-butyl-2-iodoacetamide (N-t-butyliodoacetamide) and 2-iodo-N-phenylacetamide (iodoacetanilide), are described. N-t-Butyliodoacetamide and iodoacetanilide were synthesised to purity in their d(0)-light and in their respective d(9)- and d(5)-heavy forms. The newly synthesised reagents are covalently bound to peptides containing cysteines via an alkylation reaction. The mass differences of 5 and 9 Da avoid possible problems of overlapping isotope distribution. For each alkylated cysteine a peptide mass increases, respectively, by a multiple of 113 and 133 Da for the d(0)-light form of N-t-butyliodoacetamide and iodoacetanilide. These reagents can therefore replace common alkylating reagents in existing proteomics-based applications. Alkylated peptides increase in mass in the same mass range as amino acids and remain suitable for tandem mass spectrometry (MS/MS) data acquisition and analysis. The compounds are simple to use and derivatisation is based on widely applied alkylating procedures. Preliminary results show that these reagents can be applied for both protein quantitation and identification by peptide mass finger printing and/or MS/MS techniques. Using these chemicals and the suggested workflow enables the quantitative analysis of the whole protein sample and realises access to peptides that may contain potential post-translational modifications. Other approaches that incorporate a matrix-assisted laser desorption/ionisation (MALDI) interface prior to MS can take advantage of these chemicals, such as the molecular scanner.     

Synthesis of pyrene—acridine bis-intercalators and effects of binding to DNA

A bis-intercalating compound containing pyrene and 9-aminoacridine chromophores (N-(5-(1-pyrenyl)-pentyl)-6-(9-acridinylamino) hexylamide, I), was prepared and its interaction with double-stranded DNA was investigated. Homologous compounds in which the two chromophores were connected by a linear carbon chain (pentamethylene (II), tetramethylene (III) and methylene (IV)) were also prepared. In acetonitrile solutions of the free ligands, the presence of the proximal pyrene results in reduced acridine fluorescence relative to 9methylaminoacridine (9-MAA), and the degree of quenching increases with decreasing chain length. The quenching process is assigned to exothermic electron transfer from pyrene to the excited 9-aminoacridine (9-AA) chromophore. In the presence of DNA, the relative quenching order is reversed, and I and IV are quenched more strongly than II and III. From linear dichroism experiments, it is concluded that I binds by bis-intercalation of the pyrene and acridine moieties, III and IV undergo intercalation of the acridine chromophore and II binds by partial bis-intercalation at two contiguous sites.     

The Dynamics of Populating The Lowest Triplet State Of Benzophenone Following Singlet Excitation

Measurements of the build-up of triplet-triplet absorption at 5300 A have been made during and subsequent to the excitation of benzophenone by 5 psec pulses of 3545 A radiation (frequency tripled Nd³⁺: glass fundamental). The data were fitted to a simple model that included the finite gaussian pulse width and results for the assumed process S_i

T₁ are: K^?1=16.5±3 psec for benzophenone in ethanol; k^?1 = 30 ±5 psec for benzophenone in benzene. This new type of solvent effect on triplet build-up is discussed.     

The use of fluorescence quenchers in resonance Raman spectroscopy

We discuss methods of increasing the resonance Raman signal to noise in the presence of a strong fluorescence background. By considering the time dependence of the competing processes, we conclude that for many commonly encountered systems the Raman signal can be enhanced over the fluorescence by introducing a quencher that decreases the lifetime of the radiative electronic state. The efficacy of this approach is demonstrated by the appearance of resonance Raman from fluorescein in water with the progressive addition of KI.     

Nachweis und pr?parative Trennung von Proteinaseinhibitoren und von Proteinasen mit Hilfe wasserunl?slicher Enzym- bzw. Inhibitorharze

Zusammenfassung Die Bestimmung von Inhibitoren für die Proteinasen Trypsin, Chymotrypsin, Plasmin, Thrombin und für Kallikreine in Gewebsextrakten und Körperflüssigkeiten wird diskutiert. Mit Hilfe wasserunlöslicher Proteinaseharze lassen sich Inhibitoren dieser Enzyme auf einfache und spezifische Weise aus den Extrakten isolieren. Die Aminosäurezusammensetzung der aus Pankreas von Rind, Schwein und Hund isolierten spezifischen Trypsininhibitoren wird angegeben. In ähnlicher Weise eignen sich auch wasserunlösliche Inhibitorharze zur Isolierung von Enzymen und zur einfachen Trennung von Proteinasegemischen.

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Identification and preparative separation of proteinase inhibitors and of proteinases by means of water-insoluble enzyme and inhibitor resins

Estimation procedures for inhibitors of the proteinases trypsin, chymotrypsin, plasmin (fibrinolysin), thrombin and kallikreins in extracts of organs and body fluids are discussed. Best results are obtained with synthetic substrates, e.g. N-benzoyl-arginine-p-nitroanilide (for trypsin), N-(3-carboxypropionyl)-l-phenylalanine-p-nitroanilide (for chymotrypsin) and N-benzoyl-argininethylester (for plasmin, thrombin, kallikreins). Water-insoluble resins (copolymers of ethylene and maleic acid) of proteinases are very suitable for the specific isolation of proteinase inhibitors from crude extracts of plants and organs. Inhibitors of relative low molecular weight (near 6,000) are bound in neutral buffer solutions in complex form to the proteinase, which is covalently fixed to the polyanionic carrier. Inhibitors of high molecular weights and low isoelectric points (below 6) are bound only to the fixed proteinase if the polyanionic character of the resin is neutralized. Thus we succeeded in isolating, e.g., inhibitors from soyabeans and egg white. The inhibitors are dissociated from the proteinase resins in acidic salt solutions and in urea or guanidine solutions. Water-insoluble resins of the trypsin-kallikrein inhibitor from beef organs are suitable for the isolation of trypsin, chymotrypsin and kallikreins in a similar manner.

     

Two-dimensional infrared spectroscopy of the alanine dipeptide in aqueous solution

The linear-infrared and two-dimensional infrared (2D IR) spectra in the amide-I' region of the alanine dipeptide and its (13)C isotopomers in aqueous solution (D(2)O) are reported. The two amide-I' IR transitions have been assigned unambiguously by using (13)C isotopic substitution of the carbonyl group; the amide unit at the acetyl end shows a lower transition frequency in the unlabeled species. The ratio of their transition dipole strengths remains almost unchanged upon (13)C substitution, indicating the absence of intensity transfer between two vibrators. The 2D IR cross peaks directly associated with intramode coupling in this case show a small off-diagonal anharmonicity (0.2 +/- 0.2 cm(-1)), leading to a small coupling constant (1.5 +/- 0.5 cm(-1)). The coupling and the 2D IR spectra in two different polarizations (zzzz and zxxz) are as expected for a polyproline-II (PP(II))-like conformation for dialanine, with the backbone dihedral angles (phi, psi) determined to be in the range of (-70 degrees +/- 25 degrees, +120 degrees +/- 25 degrees). Ab initio DFT calculations and normal mode decoupling analysis in the Ramachandran subspace in the neighborhood of PP(II) conformation confirm the presence of a region where the coupling is vanishingly small and support these experimental findings. The relationship between the coupling and off-diagonal anharmonicity is consolidated by examining the distribution of the latter from an ensemble averaged Hamiltonian incorporating uncorrelated diagonal frequency distributions and a small coupling (<2 cm(-1)); it is found that the most probable value for the off-diagonal anharmonicity falls into the range of experimental observations. Further, incorporating DFT results, the simulated linear-IR and 2D IR can reproduce the essential features of the measurements, including the transition frequency positions and apparent peak intensities. All the experimental results and simulations are consistent with a PP(II)-like conformation for the alanine dipeptide in aqueous solution, in which two amide-I' modes are highly localized and whose frequency distributions are uncorrelated.     

Human liver protein map: a reference database established by microsequencing and gel comparison.

This publication establishes a reference human liver protein map obtained with immobilized pH gradients. By microsequencing, 57 spots or 42 polypeptide chains were identified. By protein map comparison and matching (liver, red blood cell and plasma sample maps), 8 additional proteins were identified. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing a human liver two-dimensional gel database. This reference map can be used to identify protein spots on other samples such as rectal cancer biopsies.