Homogeneous immunoassays

Applied Biochemistry and Biotechnology -     

Oxobenzo[f]benzopyrans as new fluorescent photolabile protecting groups for the carboxylic function

The properties of three oxobenzo[f]benzopyrans as new fluorogenic photolabile protecting groups for the carboxylic function of amino acids were studied. Fluorescent amino acid conjugates were efficiently prepared and characterised. Photodeprotection of these compounds was carried out by irradiation at 300, 350 and 419 nm, the most suitable wavelength being 350 nm, on account of short irradiation times and good deprotection yields.     

The use of metallocenic esters of n-hydroxysuccinimide for metallohapten synthesis

Different organometallic markers have been described in a new technique for the labelling of many drugs. Thus metallocenic esters of [M = (;CO)₃CrC₆H₅?; (;CO)₃CrC₆H₅?(;CH₂)₃?; η-C₅H₅?FeC₅H₄?; (;CO)₃MnηC₅H₄?; (;CO)₃Mn?ηC₅H₄COCH₂CH₂?; ηC₅H₄(;ηC₅H₅)Co⁺PF^?₆] react with primary or secondary amine drugs [DRUG?NHR] for a psychostimulant drug: amphetamine; tricyclic antidepressants—desipramine and nortriptyline; a vasodilator—histamine; an adrenergic substance—norfenefrine; and for a central stimulant—meth-amphetamine, to give the metallohaptens MCON(;R)—DRUG. All these compounds have been fully characterized by different analytical methods and have potentialities for biological assays. This synthetic route was found better than one presented previously which utilized the metallocenic acid chloride MCOCI as intermediate, and could be proposed as a general synthetic route for labelling biological compounds which possess an amino group.     

Application of isotopic labeling,and gas chromatography mass spectrometry,to understanding degradation products and pathways in the thermal-oxidative aging of Nylon 6.6

Nylon 6.6 containing ¹³C isotopic labels at specific positions along the macromolecular backbone has been subjected to extensive thermal-oxidative aging at 138 °C for time periods up to 243 days. In complementary experiments, unlabeled Nylon 6.6 was subjected to the same aging conditions under an atmosphere of ¹⁸O₂. Volatile organic degradation products were analyzed by cryofocusing gas chromatography mass spectrometry (cryo-GC/MS) to identify the isotopic labeling. The labeling results, combined with basic considerations of free radical reaction chemistry, provided insights to the origin of degradation species, with respect to the macromolecular structure. A number of inferences on chemical mechanisms were drawn, based on 1) the presence (or absence) of the isotopic labels in the various products, 2) the location of the isotope within the product molecule, and 3) the relative abundance of products as indicated by large differences in peak intensities in the gas chromatogram. The overall degradation results can be understood in terms of free radical pathways originating from initial attacks on three different positions along the nylon chain which include hydrogen abstraction from: the (CH₂) group adjacent to the nitrogen atom, at the (CH₂) adjacent the carbonyl group, and direct radical attack on the carbonyl. Understanding the pathways which lead to Nylon 6.6 degradation ultimately provides new insight into changes that can be leveraged to detect and reduce early aging and minimize problems associated with material degradation.     

Specificity of Cyanine Dye L-21 Aggregation in Solutions with Nucleic Acids

Optical spectroscopy experiments were used to study the features of cyanine dye 3,3′-dimethyl-9-(2-thienyl)-thiacarbocyanine iodide (L-21) aggregation in binary solutions DMF:Tris–HCl buffer (pH = 8) containing nucleic acids (DNA or RNA). The appearance of absorption and luminescence bands associated with J-aggregates and dimers that are formed within the minor groove of DNA has been observed. The model of L-21 J-aggregate structure is proposed. It has been found that dimers are the building blocks of L-21 J-aggregates. Disorientation in dimers caused by the minor groove curvature is reason of observation of Davydov splitting in absorption spectrum of L-21 J-aggregates. In the solution containing DNA the absorption and luminescence bands of L-21 J-aggregates exhibit the specific properties that allows the dye L-21 to be used as a fluorescent probe for DNA detection.     

An Improved Plotting Package Applied to Chemical Graphing Problems

Abstract

A plotting package, written in LINC assembly language, is described. It produces high quality graphs and labels the graphs for direct submission to professional journals. A μ-LINC (Laboratory INstrument (Computer) is used to compute least-squares fitted curves and lines from data entered either on-or off-line and to draw the graphs directly on an incremental plotter. Bar graphs, line graphs, and fitted data can be plotted with options to suppress the lines and/or the points.     

Determination of T1-spin-lattice relaxation time in a two-level system by continuous wave multiquantum electron paramagnetic resonance spectroscopy in a presence of tetrachromatic microwave irradiation

Applicability of continuous wave multiquantum EPR methods to study relaxation times at X-band is examined. Multiquantum transitions excited in a two-level system by tetrachromatic irradiation are used for these studies. The Bloch equation model is applied to simulate lineshapes of the three quantum transitions as a function of frequency difference between exciting fields. The dependence of multiquantum transition signals on relaxation times and microwave amplitude is shown. On this basis a method of deducing relaxation times from these signals is formulated. The case of a homogeneously and inhomogeneously broadened resonance line is considered. Two experimental methods are used to verify the proposed hypothesis: the X-band continuous wave multiquantum EPR with four frequencies microwave field and saturation recovery EPR. The values of T₁ obtained from CW MQ EPR and SR EPR are compared.     

A Spirocyclohexyl Nitroxide Amino Acid Spin Label for Pulsed EPR Spectroscopy Distance Measurements

Site‐directed spin labeling and EPR spectroscopy offer accurate, sensitive tools for the characterization of structure and function of macromolecules and their assemblies. A new rigid spin label, spirocyclohexyl nitroxide α‐amino acid and its N‐(9‐fluorenylmethoxycarbonyl) derivative, have been synthesized, which exhibit slow enough spin‐echo dephasing to permit accurate distance measurements by pulsed EPR spectroscopy at temperatures up to 125 K in 1:1 water/glycerol and at higher temperatures in matrices with higher glass transition temperatures. Distance measurements in the liquid nitrogen temperature range are less expensive than those that require liquid helium, which will greatly facilitate applications of pulsed EPR spectroscopy to the study of structure and conformation of peptides and proteins.