Formation Of Fluorescent Compounds Research Articles

Non-tryptophan fluorescence and high molecular weight protein formation in lens crystallins of rats with chronic galactosemia: Prevention by the aldose reductase inhibitor sorbinil

Crystallin-bound non-tryptophan fluorescence and protein cross-linking were studied in chronic galactosemia. Fluorescence (excitation/emission— 370 440 nm) was significantly higher in galactosemic rats compared to controls ( P < 0·001). Accumulation of fluorescence was significantly reduced both in water soluble ( P < 0·001) and insoluble ( P < 0·005) lens fractions in galactosemic rats receiving the aldose reductase inhibitor sorbinil. High performance liquid chromatography of water soluble lens crystallins showed an increase in the content of high molecular weight proteins and the fluorescence associated with it. Sorbinil partly prevented the formation of such fluorescent high molecular weight proteins. Under reducing conditions, sodium dodecyl sulfate polyacrylamide gel electrophoresis of water soluble proteins revealed a distinct high molecular weight protein of 60 kDa in galactosemia. Sorbinil treatment completely abolished the formation of this protein. Western blotting using rabbit antiserum to bovine α-, β- and γ-crystallins revealed the presence of gamma, but not α- and β-crystallins in the 60-kDa protein. Formation of this protein may result from trimerization of γ-crystallins or from an association of γ-crystallin with a non-crystallin cytosolic protein or mixed protein cross-linking of γ-crystallins with membrane protein(s). The present study shows that polyol pathway in cataractogenesis also extends to protein cross-linking and formation of fluorescent compounds, the nature of which remains to be elucidated.

Formation of fluorescent products in the reaction of butyraldehyde and methylamine as a model of the reaction of oxidized lipids and proteins.

Formation of fluorescent compounds in the reaction of butyraldehyde and methylamine at pH 7 and 37 o C was investigated as a model of the reaction of oxidized lipids and proteins. The reaction mixture was treated with borohydride for isolation of the products. Some fluorescent products remained unchanged and the other changed into other fluorescent compounds. One of the fluorescent compounds produced on borohydride treatment was identified as 2,4-diethyl-2,5-dihydrofuran, which exhibited fluorescence with an excitation maximum at 345 nm and an emission maximum at 406 nm. The product may be derived by the condensation of 2 molecules of butyraldehyde in the presence of methylamine

Presence of nontryptophan fluorophores specifically bound to γ2-crystallin

α-, β-, and γ-crystallins have been purified from nonpathological lenses of calves. The pure proteins have been examined for nontryptophan fluorescence and fluorescent compounds have been found specifically bound to γ 2-crystallin. The protein has been unfolded by 6 m guanidine hydrochloride (Gdn-HCl) and a separation of the fluorescent compounds has been obtained by gel chromatography in the presence of 6 m Gdn-HCl. The spectroscopic features (absorbance, fluorescence) of the protein returned to normal following removal of the chromophores. The low-molecular-weight separated fluorescent compounds have been fractionated and extracted from the Gdn-HCl solution by ethyl acetate. TLC chromatography has shown the presence of kynurenine, 3-OH-kynurenine, and free tryptophan. These data suggest that direct involvement of the intrinsic protein tryptophans in the photochemical processes leading to formation of fluorescent compounds has to be excluded. Free tryptophan and intrinsic metabolic factors are probably more relevant in determining the cataractous insult.

In vivo lipid peroxidation in rat brain following intracortical Fe2+ injection.

Cerebral contusion, cortical laceration, intracerebral hematoma formation, and hemorrhagic cortical infarction cause extravasation of red blood cells, followed by hemolysis, decompartmentalization of iron, formation and deposition of hemosiderin, and an increased incidence of epilepsy. In this experiment, 10 microliter of an aqueous solution containing 100 mmol/L FeCl2, 100 mmol/L CoCl2, or 0.9% (wt/vol) NaCl were injected at a depth of 1.8 mm into rat isocortex. The rate of formation of fluorescent compounds was measured in chloroform-methanol extracts of isocortical homogenates. Significant increases in the quantity of fluorescent products of lipid peroxidation were found 120 min after the injection of 100 mmol/L FeCl2. Cobaltous chloride and saline injection had no effect on the levels of fluorescent products found in the cortical homogenates. Although the intracortical deposition of aqueous solutions containing CoCl2 or FeCl2 in rodent cortex causes acute epileptiform discharges, the epileptogenic effect of CoCl2 is transient, while the injection of iron salts causes persistent seizures. Since CoCl2 injection failed to cause formation of lipid peroxidation products while the isocortical injection of iron caused significant increase in fluorescence within the injected hemisphere, we suggest that the occurrence of iron-induced lipid peroxidation may be of importance in initiation of recurrent seizures in the rat.

7-Chloro-4-nitrobenzeno-2-oxa-1,3-diazole actin as a probe for actin polymerization.

Lysine 372 of N-ethylmaleimide actin was specifically (60%) labeled by 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole chloride (NBD-Cl), which also reacted with lysines on cyanogen bromide fragment 17 (20%) and other undetermined residues (20%). Isolation of N-ethylmaleimide peptides and two-dimensional peptide mapping demonstrated that 90% of bound N-ethylmaleimide was attached to an adjacent residue, cysteine 373, independent of the polymerization state of actin during the labeling reaction. Formation of NBD cysteine severely inhibited lysine modification. After N-ethylmaleimide blockage of cysteine 373, lysine labeling with NBD was greatly accelerated. The kinetics of formation of fluorescent compounds were biphasic, with fluorescence decreasing upon prolonged incubation of actin in NBD-Cl. Lysine 372 of purified NBD actin reproducibly responded to polymerization by a 2.2- to 2.3-fold enhancement of fluorescence. By contrast, interaction of NBD actin with several actin-binding proteins caused only very small or undetectable changes in fluorescence intensity: 10% enhancement on myosin subfragment 1 binding, about 6% quenching by DNase I, and no change at all by tropomyosin-troponin. Despite its sensitivity to polymerization the probe did not affect it. Native and modified actin polymerized randomly indicating that the rate constants for polymerization remained the same. Labeling actin with NBD did not diminish its cofactor activity for myosin ATPase activity. Contrary to previous reports we observed that myosin subfragment 1 (single myosin heads) caused actin polymerization in the absence of salt.

A new reaction in histochemistry: structure and mechanism of formation of fluorescent compounds in the reaction of tryptamine and carboxyl substituted tetrahydro- -carbolines with glyoxylic acid.

Die Identifizierung und der Bildungsmechanismus des fluoreszierenden Produktes aus Glyoxylsäure und Tryptamin wird beschrieben. Eine intramolekulare säure-katalysierte Reaktion wird für die Bildung des fluoreszierenden Produktes vorgeschlagen.