Eosin Isothiocyanate Research Articles

Plasma Membrane Calcium Pump (PMCA) Differential Exposure of Hydrophobic Domains after Calmodulin and Phosphatidic Acid Activation

The exposure of the plasma membrane calcium pump (PMCA) to the surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16 to the protein. In the presence of Ca(2+) both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [(125)I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in three main fragments: N, which includes transmembrane segments M1 and M2; M, which includes M3 and M4; and C, which includes M5 to M10. CaM decreased the level of incorporation of [(125)I]TID-PC/16 to fragments M and C, whereas phosphatidic acid decreased the incorporation of [(125)I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced by CaM and PA. To verify this point, we measured resonance energy transfer between PMCA labeled with eosin isothiocyanate at the ATP-binding site and the phospholipid RhoPE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these two probes, whereas PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.

Characterization of redox state and reductase activity of protein disulfide isomerase under different redox environments using a sensitive fluorescent assay

In this study, dieosin glutathione disulfide (Di-E-GSSG) was synthesized by the reaction of eosin isothiocyanate with GSSG. Di-E-GSSG had low fluorescence which increased ∼ 70-fold on reduction of its disulfide bond. The substrate was used to monitor the disulfide reductase activity of PDI. Di-E-GSSG is the most sensitive pseudo substrate for PDI reductase activity reported to date. This probe was further used as an analytical reagent to develop an end point assay for measuring the redox state of PDI. The reduction of Di-E-GSSG by reduced enzyme was studied in the absence of reducing agents and the redox state of PDI was monitored as a function of the stoichiometric changes in the amount of eosin-glutathione (EGSH) generated by the active-site dithiols of PDI. The redox state of PDI was also studied under variable [GSH]/[GSSG] ratios. The results indicate that PDI is in ∼ 1/2-reduced state where the [GSH]/[GSSG] ratio is between 1:1 and 3:1, conditions similar to the lumen of endoplasmic reticulum or in the extracellular environment. On the other hand, [GSH]/[GSSG] ratios of ≥ 8:1, such as in cytosol, all active-site thiols would be reduced. The study was extended to utilize Di-E-GSSG to investigate the effect of variable redox ratios on the platelet surface PDI reductase activity.

Inter-active-site distance and solution dynamics of a bivalent-bispecific single-chain antibody molecule.

The solution dynamics of a bivalent bispecific single-chain antibody (BiSCA) specific against fluorescein (Fl) and single-stranded DNA (ssDNA) were investigated. Fluorescence resonance energy transfer (FRET) studies were performed in order to estimate the average distances, R, between the anti-Fl and the anti-ssDNA active sites. In separate experiments, either 2-(dimethylamino)naphthalene-5-sulfonyl chloride coupled to the 5' end of an oligothymidylate polymer of 6 residues length (2,5-DNS-dT6) served as energy donor to Fl or eosin isothiocyanate coupled to the 5' end of an oligothymidylate polymer of 6 residues length (eosin-dT6) served as energy acceptor from Fl. Labeling of dT6 with 2,5-DNS or eosin did not significantly interfere with recognition by the anti-ssDNA binding site. With the 2,5-DNS/Fl energy transfer pair, the calculated values of R(k2 = 2/3), R(min), and R(max) were 44, 37, and 54 A, respectively. With Fl/eosin (opposite direction of FRET), values of 40, 33, and 51 A, respectively, were obtained. Considering the sizes of the two SCA domains and the length of the interdomain polypeptide linker, an R value of approximately 140 A would be expected for the extended molecule. The fact that measured R distances were on average 3-fold shorter than 140 A indicated that BiSCA was not an extended and rigid molecule. The efficiency of energy transfer increased with increasing temperature in the range of 10-30 degrees C, suggesting that conformational fluctuations of the protein resulted in decreased average distance between BiSCA active sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of Glutathione Reductase by Light: A Novel Approach to Design Redox Photo‐Enzymes

Light‐induced electron transfer within a protein, coupled with chemical transformations, was achieved with the glutathione reductase modified by eosin isothiocyanate. The dye functions both as an initiator for the electron transfer and as a relay unit in the protein backbone that facilitates the electrical communication with the redox center at which the oxidized glutathione is reduced.

Class I major histocompatibility complex antigens are not associated with the LH/CG receptor on ovine luteal cells

We have examined the rotational dynamics of the luteinizing hormone (LH) receptor on day 10 intact ovine small luteal cells and isolated plasma membranes using polarized fluorescence depletion (PFD). This technique measures rotational correlation times which are proportional to the in-membrane volume of a protein and are useful for examining changes in protein size due to receptor aggregation or protein-protein interactions. Eosin isothiocyanate (EITC)-derivatized ovine LH (EITC-oLH) bound to the LH receptor on luteal cell plasma membranes had a rotational correlation time of 20 ± 6 μs, while that for EITC-human chorionic gonadotropin (EITC-hCG)-occupied LH receptors was 46 ± 13 μs. Slower rotational times for EITC-oLH and EITC-hCG, 63 ± 19 and 87 ± 20 μs, respectively, were obtained on intact ovine luteal cells. These results indicate that the LH receptor exists as a larger molecular mass complex when binding hCG than oLH, a difference which could be attributable to hCG-induced LH-receptor interaction with additional membrane protein(s). One candidate protein for such an interaction is the Major Histocompatibility Complex (MHC) Class-I antigen. However, the rotational correlation time of EITC-anti-MHC Class-I antibody (SBU I) Fab fragments was 247 ± 34 μs, indicating that MHC Class I is located in complexes larger than those identified by EITC-OLH or EITC-hCG. Preincubation of plasma membranes with 1 nM unlabeled oLH or hCG had no significant effect on this rotational correlation time. Further, treatment of cells with SBU I had no affect on either basal or oLH-stimulated progesterone secretion. Thus it appears that the ovine luteal LH-receptor is not associated with MHC Class I and that antibody-induced aggregation of MHC Class I does not cause an LH-mimetic response.

Lipid effects on aggregation of pulmonary surfactant protein SP-C studied by fluorescence energy transfer.

The self-association of pulmonary surfactant protein SP-C in lipid vesicles was studied using fluorescence energy transfer. Bovine SP-C was labeled with two fluorescent probes, succinimidyl 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoate and eosin isothiocyanate, on the amino terminus of the protein, producing NBD-SP-C and EITC-SP-C, respectively. The N-terminus of SP-C was relatively immobile between 20 and 37 degrees C, as demonstrated by high fluorescence anisotropy of NBD-SP-C and EITC-SP-C. The mobility increased at the transition of the lipid to the fluid phase. Using fluorescence energy transfer, with NBD-SP-C as the donor and EITC-SP-C as the acceptor, a high degree of SP-C/SP-C association was found below 25 degrees C, decreasing to very little self-association above 42 degrees C in 7:1 1,2-dipalmitoylphosphatidylcholine-1,2-dipalmitoylphosphatidylglycerol (DPPC-DPPG) vesicles. The fraction of SP-C aggregated below 37 degrees C in 7:1 DPPC-DPPG was estimated from the observed energy transfer to be more than 70% of total SP-C. In various lipid mixtures, self-association of SP-C was dependent on the presence of at least some gel-phase lipids. In a lipid mixture resembling pulmonary surfactant, gradually increasing self-association was observed below 38 degrees C. The relation of the present data to the state of aggregation of SP-C in pulmonary surfactant is discussed.

Characterization of the Activation of Membrane-Bound and Soluble CF1 by Thioredoxin

The activation of spinach (Spinacia oleracea) chloroplast coupling factor 1 (CF(1)) by thioredoxin (ThR) was characterized using membrane-bound and soluble CF(1). Light generates an electrochemical proton gradient across the thylakoid membrane, which increases the accessibility of the disulfide bond on the gamma-subunit of CF(1) to reduced ThR. The proton gradient substantially accelerates the activation of CF(1) compared with thylakoids incubated in the dark with similar concentrations of dithiothreitol and ThR. The interaction of soluble CF(1) with ThR was studied using fluorescent probes. CF(1) in solution, with and without its associated epsilon-subunit, was labeled at Cys-322 of the gamma-subunit with fluoresceinyl maleimide. ThR from Escherichia coli was labeled with eosin isothiocyanate. Labeled ThR and CF(1) showed normal activities. Fluorescence energy transfer between donor fluoresceinyl maleimide and acceptor eosin isothiocyanate, manifested by a quenching of the donor fluorescence, was detected, suggesting that ThR and CF(1) form an intermolecular complex. When the epsilon-subunit was absent, quenching of donor fluorescence was approximately doubled, indicating that labeled ThR could approach more closely to the gamma-subunit of CF(1). The distance between the fluorescent probes on CF(1) and ThR was calculated to be approximately 65 A when epsilon-subunit was present and 52 A when epsilon was absent. These values are consistent with other distance measurements and energy transfer values reported previously for fluorescent probes on CF(1). Whereas the extent of quenching increased by removal of the epsilon-subunit, the apparent dissociation constant was unchanged. The quenching effect was reversed when the epsilon-subunit was added back to the titration mixture. Similarly, the addition of unlabeled ThR decreased donor quenching.

Determining the extent of labeling for tetramethylrhodamine protein conjugates

A new, relatively simple, spectrophotometric technique has been developed which is useful for accurately determining the extent of chromophore labeling of proteins. Often the absorbance spectra and extinction coefficients of dye/protein conjugates are strongly affected by changes in the chromophore microenvironment that may occur at high dye/protein ratios. In the method being presented, the microenvironment effects have been significantly reduced by denaturing the dye/protein complex in 6 M guanidine hydrochloride prior to making the necessary spectrophotometric measurements. With this approach, extinction coefficients were obtained under native and denatured conditions for tetramethylrhodamine isothiocyanate (TRITC) when bound to a model protein receptor, the sugar binding protein concanavalin A (ConA). The extinction coefficients used for TRITC/ConA conjugates under native and denaturing conditions were 6.52 × 10 4 M −1 cm −1 and 6.96 × 10 4 M −1 cm −1, respectively. These values were obtained from a model dye complex formed between TRITC and ϵ-amino- n-caproic acid which closely resembles the sidechain of lysine residues. Additional dye/ConA conjugates were prepared with tetramethylrhodamine succinimidyl ester (RHS) and eosin isothiocyanate (EITC), and the effects of microenvironment changes on these conjugates were examined. Extinction coefficients for these dyes in native and denaturing conditions, as a function of the degree of labeling, were not appreciably different indicating that changes in the microenvironment did not have a significant affect on the spectral properties of these two dyes. In summary, with this new approach it is quite easy to accurately determine the dye/protein ratio for TRITC conjugates. Also, it is expected that RHS would be a better dye than TRITC for protein conjugation because more accurate values for dye/protein ratios can be obtained under native conditions.

Potentiation of hyperthermia-induced haemolysis of human erythrocytes by photodynamic treatment. Evidence for the involvement of the anion transporter in this synergistic interaction

Heat treatment of human erythrocytes led to increased passive cation permeability, followed by haemolysis. K+ leakage was linear up to a loss of about 80% in the temperature range 46-54 degrees C. Kinetic analysis of the results revealed an activation energy of 246 kJ/mol, implicating a transition in the membrane as critical step. Pretreatment of erythrocytes with 4,4'-di-isothiocyano-2,2'-stilbenedisulphonate, chymotrypsin or chlorpromazine caused a potentiation of subsequent heat-induced K+ leakage. Photodynamic treatment of erythrocytes with Photofrin II, eosin isothiocyanate or a porphyrin-Cu2+ complex as sensitizer also induced an increase in passive cation permeability, ultimately resulting in colloid osmotic haemolysis. The combination of photodynamic treatment immediately followed by hyperthermia had a synergistic effect on K+ leakage. Analysis of the results by the Arrhenius equation revealed that both the activation energy and the frequency factor of heat-induced K+ leakage were decreased significantly by preceding photodynamic treatment, suggesting that hyperthermia and photodynamic treatment have a common target for the induction of K+ leakage. Several lines of reasoning indicate that this common target is band 3. A model is thus proposed for the observed potentiation of hyperthermically induced K+ leakage by photodynamic treatment, in which photo-oxidation of band 3 results in increased sensitivity to subsequent thermal denaturation. These phenomena may be of more general significance, as photodynamic treatment and hyperthermia interacted synergistically with respect to K+ leakage with L929 fibroblasts also.

Comparison of epidermal growth factor (EGF) receptor-receptor interactions in intact A431 cells and isolated plasma membranes. Large scale receptor micro-aggregation is not detected during EGF-stimulated early events

We have used resonance energy transfer to monitor epidermal growth factor (EGF) receptor micro-aggregation at the surface of intact human epidermoid carcinoma (A431) cells. EGF molecules labeled with fluorescein isothiocyanate and eosin isothiocyanate were demonstrated to bind tightly to cellsurface receptors, to elicit immediate changes in cytosolic free [Ca2+], and to undergo endocytosis. Under conditions which maintain the integrity of the cell, we observed no energy transfer between the donor fluorescein isothiocyanate-labeled EGF molecules and the acceptor eosin isothiocyanate-labeled growth factors bound to receptors. However, after disruption of cells by Dounce homogenization, a significant degree of energy transfer was observed (approximately 10-20%) with membranes, indicative of receptor aggregation. These results suggest that EGF does not cause micro-aggregation of the majority of its receptors on the surface of intact A431 cells within the time period of the early events associated with growth factor action. Moreover, it appears that the A431 cells contain some component which imparts a constraint on the ability of EGF receptors to aggregate, and that some of this component is lost upon the disruption of cells.

Fast rotation of EITC-labelled proteolipid after reconstitution of chloroplast ATP synthase in bilayer membranes

The chloroplast ATP synthase (CF 0CF 1) has been isolated and labelled with the triplet probe eosin isothiocyanate (EITC) when dissolved in detergent solution. On SDS-PAGE gels of the labelled enzyme, eosin fluorescence was observed almost exclusively in a single band with a molecular mass of about 8000 Da, subunit III of CF 0. At saturation, about 10 mol EITC/mol CF 0CF 1 were bound, which may indicate, that the complex contains 10 copies of subunit III. The CF 0CF 1 complex labelled in subunit III was incorporated into liposomes using the dialysis technique and the enzyme rotational diffusion in the liposome membrane was measured using laser-flash-induced absorption anisotropy of the protein-bound EITC. At 30°C and a lipid/protein ratio of 8 · 10 4:1 mol/mol uniaxial rotation with a correlation time of 200 ns at 30°C was observed. Considering the model of Saffman and Delbrück (Saffman, P.G. and Delbrück, M. (1975) Proc. Natl. Acad. Sci. USA 72, 3111–3113) and the viscosity of the azolectin membrane (Wagner et al. (1989) Eur. J. Biochem. 182, 165–173), this rotational correlation time corresponds to uniaxial rotation of a protein cylinder in the membrane with the radius of 1.1 nm. The rotating unit with this dimensions can easily be formed by two transmembrane helices. We therefore conclude that the proteolipid oligomer in CF 0, probably due to binding of EITC at lysine-48, dissociated into monomers. The other subunits of CF 0CF 1 apparently remained stable parts of the ATP synthase complex and associated with the liposome membrane. In a second attempt, CF 0CF 1 was labelled with EITC when bound to the thylakoid membrane. In this case, on SDS-PAGE gels of the labelled enzyme, eosin fluorescence was observed in a single band with a molecular mass of about 57 kDa, corresponding to the β subunit of CF 1. After incorporation of the β-labelled CF 0CF 1 into liposomes the rotational correlation time of the enzyme was 1.3–1.5 μs at 30°C. This rotation time is compatible with the uniaxial rotation of a ‘protein cylinder’ in the membrane with the diameter of 6.1 nm. Obviously, this rotating unit represents the entire CF 0CF 1 complex. The rotation rate of the reconstituted enzyme complex appeared to be determined mainly by the friction experienced by CF 0 within the membrane and therefore the observed ‘rotating unit’ represents almost exclusively CF 0. Addition of 2 μM venturicidin or 2 μM tributyltin, which are known to block proton flow through CF 0, to proteoliposomes containing the β-subunit-labelled CF 0CF 1 increased the rotational correlation time of the enzyme to about 2 μs. This result shows that the both inhibitors block the H + flow through CF 0CF 1 by changing the overall structure of the F 0 part. In contrast to this result, with proteoliposomes containing the subunit-III-labelled CF 0CF 1 no changes in the rotational time were observed after addition of venturicidin and tributyltin. This indicates that binding of either of the two inhibitors does not affect the conformation of the monomeric subunit III to any remarkable extent.

Visualization of Epidermal Growth Factor (EGF) Receptor Aggregation in Plasma Membranes by Fluorescence Resonance Energy Transfer: Correlation of Receptor Activation with Aggregation

Fluorescence resonance energy transfer between epidermal growth factor (EGF) molecules, labeled with fluorescent reporter groups, was used as a monitor for EGF receptor-receptor interactions in plasma membranes isolated from human epidermoid A431 cells. Epidermal growth factor molecules labeled at the amino terminus with fluorescein isothiocyanate served as donor molecules in these energy transfer measurements, while EGF molecules labeled with eosin isothiocyanate at the amino terminus served as the energy acceptors. Both of these derivatives were shown to be active in binding to membrane receptors and in the activation of the endogenous receptor/tyrosine kinase activity. We found that membranes in the absence of added metal ion activators showed relatively little energy transfer (approximately 10% donor quenching) between the labeled growth factors. However, divalent metal ion activators of the EGF receptor/tyrosine kinase caused a significant increase in the extent of energy transfer between the labeled EGF molecules. Specifically, in the presence of 20 mM MgCl2, the extent of quenching of the donor fluorescence increased to 25% (from 10% in the absence of metal), while in the presence of 4 mM MnCl2, the extent of energy transfer was increased still further to 40-50%. The addition of an excess of EDTA resulted in the reversal of the observed energy transfer to basal levels. The increased energy transfer in the presence of these divalent cations correlated well with the ability of these metals to stimulate the EGF receptor/tyrosine kinase activity. However, the extent of receptor-receptor interactions measured by energy transfer was independent of receptor autophosphorylation. Overall, these results suggest that conditions under which the EGF receptor is primed to be active as a tyrosine kinase, within a lipid milieu, result in an increased aggregation of the receptor.

Chemical modification of the active site of ferredoxin-NADP + reductase and conformation of the binary ferredoxin/ferredoxin-NADP + reductase complex in solution

Ferredoxin-NADP + reductase (EC 1.18.1.2) was chemically modified by the triplet probe eosin isothiocyanate (eosin-NES). Incorporation of 1 mol eosin-NCS/mol ferredoxin-NADP + reductase completely inhibited binding of NADP +/NADPH to the enzyme. Binding of eosin without the reactive group to the enzyme was shown to be reversible but to compete with NADP +/NADPH with a K i of approx. 5 μM. The binding site of eosin-NCS has been located in the primary sequence ferredoxin-NADP + reductase. After specific cleavage of arginine with trypsin a single labelled peptide was obtained and identified as the fragment from residue 179–228 in the primary sequence. Binding of eosin-NCS occurred in either of two predicted helices (residues 179–189 or 212–228) which are both part of an α/β structure characteristic for nucleotide binding folds. The rotational diffusion in solution of the eosin-labelled ferredoxin-NADP + reductase and its complex with ferredoxin was measured with laser flash spectroscopy under photoselection. From the measured rotational correlation times and the known structure of ferredoxin-NADP + reductase at 3.7 Å resolution, we propose that ferredoxin is bound to ferredoxin-NADP + reductase between the two domains of the flavoprotein. The two ferredoxin-NADP + reductase domains and ferredoxin form a triangle which results in a highly integrated binary complex.

Effect of phospholipid:protein ratio on the state of aggregation of the (Ca2+-Mg2+)-ATPase.

The organization of the (Ca2+-Mg2+)-ATPase has been studied in reconstituted systems by fluorescence polarization of the ATPase labeled with fluorescein isothiocyanate (FITC) and resonance energy transfer between ATPase labeled with FITC and with eosin isothiocyanate (EITC). The fluorescence polarization of FITC-ATPase was found to decrease with increasing labeling ratio FITC:ATPase, indicating depolarization as a result of resonance energy transfer between ATPase molecules. Fluorescence polarization was, however, independent of the molar ratio of phospholipid to protein above a molar ratio of 50:1. Resonance energy transfer between FITC-ATPase and EITC-ATPase was also found to be independent of phospholipid:protein ratio. It is suggested therefore that the ATPase is not randomly distributed in the plane of the membrane but rather forms ordered clusters (probably rows of monomers or dimers) on the fluorescence time scale (nanoseconds) even in the presence of a large excess of phospholipid. This organization within the membrane is dependent both on the chemical structure of the phospholipid and on its physical phase.

Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R-NH2 group, presumably the epsilon-NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. 11, 1535-1558 (1962)] provides clear evidence that a pH 8 eight R-NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non-essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s). One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isothiocyanate. In the doubly labeled protein non-radiative single-singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (+/- 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8-1.1, 1.9-2.1, 2.9-3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8-5.1 nm from the donor site and third site being 6.4 nm apart from the donor. Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein-bound dansyl chloride and eosin isothiocyanate was observed. Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate-binding site with one lysyl residue crucial for activity.

Protein rotational motion in solution measured by polarized fluorescence depletion

A microscope-based system is described for directly measuring protein rotational motion in viscous environments such as cell membranes by polarized fluorescence depletion (PFD). Proteins labeled with fluorophores having a high quantum yield for triplet formation, such as eosin isothiocyanate (EITC), are examined anaerobically in a fluorescence microscope. An acousto-optic modulator generates a several-microsecond pulse of linearly polarized light which produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. When the sample is then probed with light polarized parallel to the excitation pulse, fluorescence recovers over 0-1,000 microseconds as the sum of two exponentials. One exponential corresponds to triplet decay and the other to the rotational relaxation. An exciting pulse perpendicular to the probe beam is then applied. Fluorescence recovery following this pulse is the difference of the same two exponentials. Equations for fluorescence recovery kinetics to be expected in various experimentally significant cases are derived. Least-squares analysis using these equations then permits the triplet lifetime and rotational correlation time to be determined directly from PFD data. Instrumentation for PFD measurements is discussed that permits photobleaching recovery measurements of lateral diffusion coefficients using the same microscope system. With this apparatus, both rotational and translational diffusion coefficients (Dr, Dt) were measured for EITC-labeled bovine serum albumin in glycerol solutions. Values obtained for Dr and Dt are discussed in light of both the PFD models and the experimental system.

Chemical modification of the phospho enolpyruvate carboxylase from maize leaves and its conformation in isotropic solution. Studies via triplet lifetime and rotational diffusion using eosin isothiocyanate as label

Phospho enolpyruvate carboxylase from maize leaves was covalently labeled with the triplet probe, eosin-5-isothiocyanate. The effect of chemical modification on the enzyme activity was investigated. The enzyme-bound triplet probe was used to measure the rotational diffusion of the enzyme in isotropic solution. Modification of the enzyme with eosin isothiocyanate resulted in a loss of enzymatic activity in a time- and concentration-dependent manner. Complete inactivation could be correlated with the incorporation of 4 mol of eosin isothiocyanate/mol of enzyme. However, complete protection against inactivation could be obtained by phospho enolpyruvate plus Mg 2+. The labeled enzyme revealed a rotational correlation time of 1 μs in aqueous buffer at 20°C, either in the absence or in the presence of all substrates. When HCO 3 − and Mg 2+ or phospho enolpyruvate and Mg 2+ were present only, a rotational correlation time of 2.1 μs was obtained. These results indicate the presence of four essential ϵNH 2 groups in the enzyme, which are modulated by phospho enolpyruvate plus Mg 2+, either in their location within the protein or/and in their apparent p K (reactivity to eosin isothiocyanate). From the rotational correlation times obtained, the hydrodynamic structure of the enzyme in the abscence or presence of all substrates could be described best by a tetrahedron arrangement of four monomeric subunits each with an apparent molecular weight of 100000.

A new hypothesis for the target in photohemolysis: dimers of the band 3 protein.

Abstract— Photohemolysis has been investigated extensively during the past 20 years, but the membrane target has eluded identification. It is proposed here that the target is the dimer of the band 3 anion exchange protein. This new hypothesis is based on three observations from the literature: (1) the kinetics of photohemolysis sensitized by exogenous sensitizers or induced by UV light obey a power dependence with light dose, with an exponent near two. From target theory this implies a two‐component target. (2) Band 3 molecules exist in the erythrocyte membrane as dimers. (3) Binding of eosin to band 3 via an isothiocyano group markedly increases its potency to sensitize hemolysis. Experiments with eosin isothiocyanate bound to band 3 show that lytic rate varies as the 1.45 power of light dose, and that the bound sensitizer bleaches considerably, thereby blunting the effectiveness of higher light doses.

Rotational mobility and domain flexibility of membrane-bound bacterial coupling factor as detected with the triplet probe eosin-isothiocyanate

The membrane-bound coupling factor (BF 1) of chromatophores from the photosynthetic bacerium Rhodopseudomonas sphaeroides was covalently labeled with the triplet probe eosin-isothiocyanate. The labeled enzyme was isolated and functionally reconstituted into depleted chromatophores from the same bacterium. ATPase and ATP synthase activities of the reconstituted vesicles were strongly dependent on the labeling conditions, decreasing at increasing load of eosin molecules per BF 1. When labeling was carried out in the dark and in the presence of ATP, one molecule of eosin isothiocyanate was bound per BF 1 and the activities catalyzed by the reconstituted and labeled enzyme were as high as in untreated chromatophores. Upon light energization of the chromatophore membrane, a large conformational change of BF 1 could be detected by using the triplet probe as a spectroscopic tool. The domain flexibility and rotational mobility of the reconstituted coupling enzyme were directly related to the enhancement of the ATPase activity induced by light. Both the light-stimulated ATPase activity and conformational changes could be prevented by addition of ADP or oligomycin and affected to the same extent by uncouplers and inhibitors of electron transport. Moreover, the detected conformational changes were reversible in time, appearing with a half-time of 10 ms upon illumination of the chromatophores, and disappearing with a half-time of 70 ms in the dark. The results obtained prove the feasibility of the spectroscopic technique in detecting conformational changes of the membrane-bound BF 1, similarly to what already has been observed for chloroplast coupling factor (Wagner, R. and Junge, W. (1980) FEBS Lett. 114, 327–333), and add to the possibility of characterizing, by this method, energy transduction at a molecular level.

Measurement of the rotational behaviour of virus particles during adsorption to the host cell surface

The rotational diffusion of bacteriophage ϵ15 was measured before and after virus adsorption to outermembrane vesicles of the host Salmonella anatum. The virus capsid was labeled with eosin isothiocyanate, and the decay of transient dichroism following dye excitation by pulses of plane-polarized light was measured. From the data, the rotational diffusion constant of the unadsorbed virion and its hydrodynamic diameter were estimated and found to be consistent with electron microscopic measurements of the capsid dimensions. Addition of outer membrane vesicles of S. anatum to the virus suspension revealed the immobilization of the virus particles on the membrane surface.