Light/dark labeling differences in chloroplast membrane polypeptides associated with chloroplast coupling factor o.

The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission. Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights 0f 32 000, 23 000 and 15 000. The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF1-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark difference in labeling are observed in CF1-depleted chloroplasts which have been treated with N,N'-dicyclohexylcarbodiimide. The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CFo) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CFo, and the light/dark labeling differences may indicate conformational changes within CFo. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis.     

Asymmetric labeling of amino lipids in liposomes.

Fluorescamine and trinitrobenzenesulfonate were used as chemical probes to differentially label amino phospholipids in liposomes. At low concentrations, fluorescamine reacts primarily with amino lipids on the external half of the bilayer. Further increase in fluorescamine concentration resulted in a linear increase of labeling indicating penetration and reaction with the internal half of the bilayer. Because of the pH requirements of the fluorescamine reaction, internal labeling was eliminated with a H+ gradient: inside acidic/outside alkaline. Differential labeling was also achieved with trinitrobenzenesulfonate, which is normally not permeable but which can be transported by valinomycin-K+ complex and react with internal amines. Thus, either half of the bilayer can be labeled with the same or different reagents. When liposomes were double-labeled, the fluorescence of fluorescamine was quenched by the trinitrobenzenesulfonate label. This quenching was reversed by solubilizing the liposomes with acidic ethanol. No quenching occurred when fluorescamine-labeled liposomes were mixed with trinitrobenzenesulfonate-reacted liposomes (or trinitrophenylated methylamine) suggesting close proximity of two labels is required for quenching. Conditions which promoted vesicular fusion promptly produced quenching. These differential labeling procedures can be usefully applied to quantitate aminolipids on internal and external vesicular surface, monitor vesicular fusion, and assess liposomal structure.     

Asymmetric labeling of amino lipids in liposomes

Fluorescamine and trinitrobenzenesulfonate were used as chemical probes to differentially label amino phospholipids in liposomes. At low concentrations, fluorescamine reacts primarily with amino lipids on the external half of the bilayer. Further increase in fluorescamine concentration resulted in a linear increase of labeling indicating penetration and reaction with the internal half of the bilayer. Because of the pH requirements of the fluorescamine reaction, internal labeling was eliminated with a H⁺ gradient: inside acidic/outside alkaline. Differential labeling was also achieved with trinitrobenzenesulfonate, which is normally not permeable but which can be transported by valinomycin K⁺ complex and react with internal amines. Thus, either half of the bilayer can be labeled with the same or different reagents.When liposomes were double-labeled, the fluorescence of fluorescamine was quenched by the trinitrobenzenesulfonate label. This quenching was reversed by solubilizing the liposomes with acidic ethanol. No quenching occurred when fluorescamine-labeled liposomes were mixed with trinitrobenzenesulfonate-reacted liposomes (or trinitrophenylated methylamine) suggesting close proximity of two labels is required for quenching. Conditions which promoted vesicular fusion promptly produced quenching.These differential labeling procedures can be usefully applied to quantitate aminolipids on internal and external vesicular surfaces, monitor vesicular fusion, and assess liposomal structure.     

Light/dark labeling differences in chloroplast membrane polypeptides associated with chloroplast coupling factor 0

The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission.Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights of 32 000, 23 000 and 15 000.The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF₁-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark differences in labeling are observed in CF₁-depleted chloroplasts which have been treated with N,N′-dicyclohexylcarbodiimide.The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CF_o) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CF_o, and the light/dark labeling differences may indicate conformational changes within CF_o. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis.     

Studies on the reaction of fluorescamine with primary amines

Fluorescamine is a useful reagent for the fluorometric assay of primary amines. The extent of the reaction between fluorescamine and primary amines, as well as the fluorescence intensities of the resulting fluorophors depend on pH, solvent composition and reagent concentration. Optimum values for these variables further depend on the amine under study. The influence of these parameters on the fluorogenic reaction of representative amines, and on their fluorophoric derivatives has been investigated, and the results are reported here.     

Fluorescent labeling of proteins in sodium dodecyl sulfate complexes with fluorescamine

The effects of sodium dodecyl sulfate on the fluorescent labeling of proteins were studied. Of 57 primary amine groups in bovine serum albumin, no more than 7 are titrable by fluorescamine. Fluorescamine labeling does not cause appreciable conformational changes of proteins. The extent of labeling of proteins decreases as the concentrations of sodium dodecyl sulfate increases. The fluorescence properties of labeled primary amine are only slightly affected by the polarities of the solvents. The inhibitory effects of sodium dodecyl sulfate upon labelings are interpreted as the low permeability of fluorescamine toward the highly charged envelopes of sodium dodecyl sulfate-protein micelles.     

Inhibition of membrane-bound succinate dehydrogenase by fluorescamine

Fluorescamine rapidly inactivated membrane-bound succinate dehydrogenase. The inhibition of the enzyme by this reagent was prevented by succinate and malonate, suggesting that the group modified by fluorescamine was located at the active site. The modification of the active site sulfhydryl group by 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) did not alter the inhibitory action of fluorescamine. However, the protective effect of malonate against fluorescamine inhibition was abolished in the enzyme modified at the thiol.     

The use of fluorescamine as a detection reagent in protein microcharacterization

With recent advances in protein microchemistry, compatible methods for the preparation and quantitation of proteins and peptides are required. Fluorescamine, a reagent which reacts with primary amino groups has been used successfully to detect amino acids, peptides, and proteins in various micromethods. This article discusses these methods which include (1) amino acid analysis of protein and peptide hydrolysates with postcolumn fluorescamine derivatization; (2) purification and characterization of proteins and peptides by reversed-phase HPLC with postcolumn fluorescamine derivatization; (3) purification of peptides by two-dimensional chromatography and electrophoresis on thin-layer cellulose with fluorescamine staining; and (4) electroblotting of protein bands from SDS-PAGE to glass fiber filters and polyvinylidene difluoride (PVDF) membranes with fluorescamine staining. In addition, this article also compares a postcolumn fluorescamine detection system with a UV detection system in the applications of amino acid analysis and reversed-phase HPLC protein/peptide analysis.     

Uncoupling of Ca2+ transport in sarcoplasmic reticulum as a result of labeling lipid amino groups and inhibition of Ca2+-ATPase activity by modification of lysine residues of the Ca2+-ATPase polypeptide

Limited labeling of amino groups with fluorescamine in fragmented sarcoplasmic reticulum vesicles inhibits Ca2+-ATPase activity and Ca2+ transport. Under the labeling conditions used, 80% of the label reacts with phosphatidylethanolamine and 20% with the Ca2+-ATPase polypeptide. This degree of labeling does not result in vesicular disruption or in loss of vesicular proteins and does not increase the membrane permeability to Ca2+. Fluorescamine labeling of a purified Ca2+-ATPase devoid of aminophospholipids also inhibits Ca2+-ATPase activity, suggesting that labeling of lysine residues of the enzyme polypeptide is responsible for the inhibition of Ca2+-ATPase activity in sarcoplasmic reticulum. Fluorescamine labeling interferes with phosphoenzyme formation and decomposition in both the native vesicles and the purified enzyme; addition of ATP during labeling, and with less effectiveness ADP or AMP, protects both partial reaction steps. Addition of a nonhydrolyzable ATP analog protects phosphoenzyme formation but not decomposition. The inhibition of Ca2+ transport but not of Ca2+-ATPase occurs in sarcoplasmic reticulum vesicles labeled in the presence of ATP, indicating that the transport reaction is uncoupled from the Ca2+-ATPase reaction. The inhibition of Ca2+ transport but not of Ca2+-ATPase activity is also found in sarcoplasmic reticulum vesicles in which only phosphatidylethanolamine has reacted with fluorescamine. Furthermore, the extent of labeling of phosphatidylethanolamine is correlated with the inhibition of Ca2+ transport rates. The inhibition of Ca2+ transport is a reflection of the inhibition of Ca2+ translocation and is not due to an increase in Ca2+ efflux. We propose that labeling of phosphatidylethanolamine perturbs the lipid environment around the enzyme, producing a specific defect in the Ca2+ translocation reaction.     

Effects of fluorescamine labeling on mitochondrial ATPase activity.

Fluorescamine labeling of rat liver mitochondria enhances the ATPase activity. It reached maximum stimulation when mitochondria were treated with 30–34 nmol fluorescamine per mg of mitochondrial protein. This stimulation is inhibited by N,N′-dicyclohexylcarbodiimide. The maximum stimulation caused by labeling is the same as that obtained from uncoupler with optimum concentration. The chemiosmotic potential (Δμ_H⁺) decreases as the labeling increased. However, Δμ_H⁺ is not abolished completely even when ATPase activity reaches a maximum. The results suggest that primary amino groups may be involved in controlling mitochondrial ATPase activity.     

The transbilayer distribution of phosphatidylethanolamine in erythroid plasma membranes during erythropoiesis

Fluorescamine was used to assess the transbilayer distribution of phosphatidylethanolamine in the plasma membrane of murine erythroid progenitor cells, CFU-E (colony-forming unit erythroid), at different stages of their differentiation pathway. Intact cells were exposed to increasing concentrations of fluorescamine and the amount of labeled phosphatidylethanolamine was determined by measuring the fluorescence intensity of its fluorescamine derivative. A semilogarithmic plot of the dose-response curve revealed three different pools of phosphatidylethanolamine, representing its fractions in, respectively, the inner- and outer monolayers of the plasma membrane and subcellular membrane systems. These results show that 9-11% of the total cellular phosphatidylethanolamine is present in the outer leaflet and 9-10% of it is located in the inner leaflet of the plasma membrane in early as well as late erythroblasts. This symmetric distribution of phosphatidylethanolamine over the two halves of the bilayer in the plasma membrane of CFU-E is very similar to that observed earlier in the plasma membrane of friend erythroleukaemic cells (Rawyler, Van der Schaft, Roelofsen and Op den Kamp (1985) Biochemistry 24, 1777-1783). These observations imply that the characteristic asymmetric distribution of phosphatidylethanolamine, as is found in mature erythrocytes, is accomplished at a very late stage of erythropoiesis and possibly during enucleation of the cells or shortly thereafter.     

The fluorescence of fluorescamine-amino acids.

Fluorescamine reacts with various amino acids to yield solutions exhibiting different amounts of fluorescence, and the fluorogenic reaction does not go to completion. To investigate these phenomena, we measured the lifetimes and quantum yields of the fluorescamine-amino acids and studied their rates of formation by stopped-flow fluorescence and transmission measurements. The quantum yields were similar (0.11 ± 0.03), as were the lifetimes (3.5 ± 0.5 ns). The only exceptions were the derivatives of tryptophan and cysteine, which were internally quenched. It was concluded that the chemical yields of the fluorescamine-amino acids varied greatly. Kinetic experiments showed wide variations in rates, with some amino acids requiring several seconds for reaction under some conditions. Proline, on the other hand, reacted rapidly, with a second order rate constant of 6.2 × 10^?4 liter mol^?1 s^?1. Sequential additions of fluorescamine to amino acids were more efficient in producing the fluorescent product than the same amount of reagent added all at once; this suggested that fluorescamine was inactivated by a concentration-dependent process. A mechanism to explain the low chemical yields is proposed in which both base and amine catalyze the inactivation of fluorescamine.     

Fluorescence probes specific for the plasma membrane of intact human erythrocytes and lymphocytes

Human erythrocytes and lymphocytes were isolated from venous blood and subjected to one of two protocols. In one protocol the suspended cells were labeled with fluorophore (fluorescamine or 12(9)AS). This procedure was followed sequentially by cellular lysis, cellular fractionation, and fluorescence and absorption readings. In the other protocol the suspended cells were lysed, and then the cellular homogenate labeled with fluorophore followed by cellular fractionation and spectroscopy readings. The lymphocytes were fractionated into plasma membrane, cytosol, and nuclear-mitochondrial fractions and the erythrocytes into plasma membrane and cytosol fractions. The results demonstrate that under the given labeling conditions, both fluorescamine and 12(9)AS are highly localized to the plasma membrane of intact human erythrocytes and lymphocytes. Furthermore, by P-31 NMR analysis, fluorophore labeling did not alter cellular high energy phosphate metabolism or cellular permeability to Mn²⁺. Therefore, these fluorophores are potentially powerful probes of human erythrocyte and lymphocyte plasma membrane dynamics in inherited and acquired disease states.     

Fluorescent labeling of the surface proteins of erythrocyte membranes using cycloheptaamylose-fluorescamine complex.

Fluorescamine was incorporated in cycloheptaamylose and the resulting complex was utilized as a new probe for fluorescent labeling of the surface proteins of erythrocytes. The complex reacted with erythrocyte membrane at 37°, pH 7 to 8 without using organic solvents. The major membrane proteins of ghosts were labeled, whereas the complex did not penetrate in the intact erythrocytes and labeled only surface proteins. Particular advantages are that the complex itself and its hydrolysis product were nonfluorescent when electrophoresed in sodium dodecyl sulfate polyacrylamide gels.     

A new fluorescent microassay method for pepsin using succinyl-albumin.

A method was developed for fluorescent microassay of pepsin with a fluorescent reagent, fluorescamine, and a nonquenching substrate, succinyl-albumin. In this method hydrolysis of succinyl-albumin by pepsin at pH 2,0 was stopped by adding phosphate buffer, pH 6.1, and newly liberated amino groups in the reaction mixture were determined quantitatively by fluorescence after adding fluorescamine. Fluorescence increased linearly with 1.0 to 18 ng of hog pepsin. The assay was 200 times more sensitive than the modified micromethod of Anson [(1939) J. Gen. Phys.22, 79–89].     

A fluorescent assay for proteolytic enzymes

A method is described which permits the assay of proteolytic enzyme activity on protein substrates without precipitation or filtration steps, utilizing a fluorescent reagent which is specific for primary amines. The assay is about 100 times more sensitive than the Lowry method, much faster and less complicated. Ambiguities concerning the absorbing species are largely eliminated. The reagent (Fluorescamine, Hoffmann-La Roche RO-20-7234) yields fluorescent compounds with amino acids at pH 9.0 and with peptides at pH 6.8, but possesses no fluorescence by itself.     

Characterization of a photosensitive glucose derivative. A photoaffinity reagent for the erythrocyte hexose transporter

The photosensitive reagent 6-N-(4-azido-2-hydroxy-3,5-diiodobenzoyl)-D-glucosamine has been assessed as a potential photoaffinity label for the hexose transporter. Under zero-trans conditions, transport experiments performed in the dark reveal that the reagent inhibits the uptake of D-glucose in resealed human erythrocyte ghosts. Increasing the concentration of glucose in the transport medium has a protective effect, reducing the inhibition. Kinetic analysis indicates that the probe acts as a competitive inhibitor with high affinity for the erythrocyte hexose transporter (Ki between 0.07 and 0.2 microM). Exposure to a 280 nm filtered high intensity mercury-vapor lamp results in a rapid and efficient photolysis. At low concentrations of the probe, specific labeling of membrane preparations was observed. Autoradiograms of 10% SDS gels revealed the specific labeling of bands 4.51 and 6. This labeling was concentration-dependent and protected by D-glucose (not the L-isomer) and phloretin in the medium. When subjected to multiple exposures of low concentration of the photoaffinity reagent, apparent saturation was achieved.     

Effect of fluorescamine modification of purple membranes on exciton coupling and light-to-dark adaptation

Purple membranes of $\text{Halobacterium}\text{,}\text{halobium}$ were modified with fluorescamine. At pH 8.8, with a molar ratio of fluorescamine to bacteriorhodopsin of 170, about 6 residues of lysine were modified while the arginines were not affected at all. Except for the appearance of the fluorescamine peak at 394 nm and some broadening of the chromophore peak at 570 nm, the absorption spectrum of bacteriorhodopsin was not significantly changed after modification. After fluorescamine modification, circular dichroism studies indicated loss of exciton coupling between bacteriorhodopsin molecules in the purple membrane. Rotational diffusion studies suggested enhanced mobility of the chromophore after modification. However, the spectral changes accompanying the light-to-dark adaptation of purple membranes were not prevented by fluorescamine modification. The implications of these findings are that exciton coupling between neighboring bacteriorhodopsin molecules in the purple membrane is not required for light-to-dark adaptation.     

Peptidyl sulfonium salts. A new class of protease inhibitors

The possibility has been examined that peptidylmethyl sulfonium salts might affinity label proteases by an alkyl transfer from sulfur to an active center residue. The synthesis of a number of agents of this type is described as well as initial results of their effect on cysteinyl proteases, papain and cathepsin B. These are readily inactivated by reagents in which the peptidyl portion contains features that promote binding to the proteases such as a penultimate phenylalanine residue. Irreversible inactivation ensues by transfer of the peptidyl portion, not methyl groups. Peptidylmethyl sulfonium salts lose a proton to form an ylide structure which may be the prevalent form at physiological pH values. The ylide may also be the active affinity labeling form of the reagent since the rate of inactivation of cathepsin B increases with pH. In contrast, the action of another affinity labeling reagent for cathepsin B, benzyloxycarbonyl-Phe-AlaCHN2, a diazomethyl ketone, is relatively independent of pH.     

The use of fluorescamine as a probe for labeling the outer surface of the plasma membrane

A rapid method was developed to label the outer surface of chick embryo fibroblasts with fluorescamine without disruption of the cell monolayer. Polyacrylamide gel electrophoresis resolved two distinct areas of fluorescence: a group of high molecular weight polypeptides and several rapidly migrating species. The latter were demonstrated by tlc to be phospholipids. Fluorescamine did not label internal components of the cell as evidenced by two intracellular proteins which were found to be non-fluorescent. Intact normal cells were labeled 3-fold more than transformed cells, indicating a possible loss of exposed sites at the surface, while disrupted cells, subsequently labeled, yielded similar amounts of fluorescence.