Heat perturbation of bovine eye lens alpha-crystallin probed by covalently attached ratiometric fluorescent dye 4'-diethylamino-3-hydroxyflavone

Bovine eye lens alpha-crystallin was covalently labeled with 6-bromomethyl-4'-diethylamino-3-hydroxyflavone and studied under native-like conditions and at the elevated temperature (60 degrees C) that is known to facilitate alpha-crystallin chaperone-like activity. This novel SH-reactive two-band ratiometric fluorescent probe is characterized by two highly emissive N*- and T*-bands; the latter appears due to excited state intramolecular proton transfer reaction. The positions of these bands and the ratio of their intensities for the alpha-crystallin-dye conjugate are the sensitive indicators of polarity of the dye environment and its participation in intermolecular hydrogen bonding. Although we found that the dye labels both the SH and the NH2 groups in alpha-crystallin, a recently developed procedure allowed us to distinguish between the heat-induced spectral changes of the dye molecules attached to SH and NH2 groups. We observed that at elevated temperature the environment of the SH-attached dye becomes more polar and flexible. The number of H-bond acceptor groups in the vicinity of the dye decreases. Since alpha-crystallin contains a single Cys residue within the C-terminal domain of its (alpha)A subunit (the (alpha)B subunit contains none), we can attribute the observed effects to temperature-induced changes in the C-terminal domain of this protein.     

A spin label that binds to myosin heads in muscle fibers with its principal axis parallel to the fiber axis.

We have used an indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione), designated InVSL, to study the orientation of myosin heads in bundles of chemically skinned rabbit psoas muscle fibers, with electron paramagnetic resonance (EPR) spectroscopy. After reversible preblocking with 5,5'-dithiobis(2-nitro-benzoic acid) (DTNB), we were able to attach most of the spin label covalently and rigidly to either Cys 707 (SH1) or Cys 697 (SH2) on myosin heads. EPR spectra of labeled fibers contained substantial contributions from both oriented and disordered populations of spin labels. Similar spectra were obtained from fibers decorated with InVSL-labeled myosin heads (subfragment 1), indicating that virtually all the spin labels in labeled fibers are on the myosin head. We specifically labeled SH2 with InVSL after reversible preblocking of the SH1 sites with 1-fluoro-2,4-dinitrobenzene (FDNB), resulting in a spectrum that indicated only disordered spin labels. Therefore, the oriented and disordered populations correspond to labels on SH1 and SH2, respectively. The spectrum of SH2-bound labels was subtracted to produce a spectrum corresponding to SH1-bound labels, which was used for further analysis. For this corrected spectrum, the angle between the fiber axis and the principal axis of the spin label was fitted well by a Gaussian distribution centered at theta o = 11 +/- 1 degree, with a full width at half-maximum of delta theta = 15 +/- 2 degrees. The unique orientation of InVSL, with its principal axis almost parallel to the fiber axis, makes it complementary to spin labels previously studied in this system. This label can provide unambiguous information about axial rotations of myosin heads, since any axial rotation of the head must be reflected in the same axial rotation of the principal axis of the probe, thus changing the hyperfine splitting. Therefore, InVSL-labeled fibers have ideal properties needed for further exploration myosin head orientation and rotational motion in muscle.     

Protein-flavonol interaction: fluorescence spectroscopic study

Recent studies have shown that various synthetic as well as therapeutically active naturally occurring flavonols possess novel luminescence properties that can potentially serve as highly sensitive monitors of their microenvironments in biologically relevant systems. We report a study on the interactions of bovine serum albumin (BSA) with the model flavonol 3-hydroxyflavone (3HF), using the excited-state proton-transfer (ESPT) luminescence of 3HF as a probe. Upon addition of BSA to the flavonoid solutions, we observe remarkable changes in the absorption, ESPT fluorescence emission and excitation profiles as well as anisotropy (r) values. Complexation of 3HF with protein results in a pronounced shift (20 nm) of the ESPT emission maximum of the probe (from lambda(max)(em) = 513 nm to lambda(max)(em) = 533 nm) accompanied by a significant increase in fluorescence intensity. The spectral data also suggest that, in addition to ESPT, the protein environment induces proton abstraction from 3HF leading to formation of anionic species in the ground state. Fairly high values of anisotropy are observed in the presence of BSA for the tautomer (r = 0.25) as well as anion (r = 0.35) species of 3HF, implying that both the species are located in motion-restricted environments of BSA molecules. Analysis of relevant spectroscopic data leads to the conclusions that two binding sites are involved in BSA-3HF interaction, and the interaction is slightly positively cooperative in nature with a similar binding constant of 1.1 - 1.3 x 10(5) M(-1) for both these sites. Proteins 2001;43:75-81.     

Simultaneous probing of hydration and polarity of lipid bilayers with 3-hydroxyflavone fluorescent dyes

The penetration of water into the hydrophobic interior leads to polarity and hydration profiles across lipid membranes which are fundamental in the maintenance of membrane architecture as well as in transport and insertion processes into the membrane. The present paper is an original attempt to evaluate simultaneously polarity and hydration properties of lipid bilayers by a fluorescence approach. We applied two 3-hydroxyflavone probes anchored in lipid bilayers at a relatively precise depth through their attached ammonium groups. They are present in two forms: either in H-bond-free form displaying a two-band emission due to an excited state intramolecular proton transfer reaction (ESIPT), or in H-bonded form displaying a single-band emission with no ESIPT. The individual emission profiles of these forms were obtained by deconvolution of the probes' fluorescence spectra. The polarity of the probe surrounding the bilayer was estimated from the two-band spectra of the H-bond-free form, while the local hydration was estimated from the relative contribution of the two forms. Our results confirm that by increasing the lipid order (phase transition from fluid to gel phase, addition of cholesterol or decrease in the lipid unsaturation), the polarity and to a lesser extent, the hydration of the bilayers decrease simultaneously. In contrast, when fluidity (i.e. lipid order) is kept invariant, increase of temperature and of bilayer curvature leads to a higher bilayer hydration with no effect on the polarity. Furthermore, no correlation was found between dipole potential and the hydration of the bilayers.     

Yeast src homology region 3 domain-binding proteins involved in bud formation

The yeast protein Bem1p, which bears two src homology region 3 (SH3) domains, is involved in cell polarization. A Rho-type GTPase, Rho3p, is involved in the maintenance of cell polarity for bud formation, and the rho3 defect is suppressed by a high dose of BEM1. Mutational analysis revealed that the second SH3 domain from the NH2 terminus (SH3-2) of Bem1p is important for the functions of Bem1p in bud formation and in the suppression of the rho3 defect. Boi2p, which bound to SH3-2 Bem1p, was identified using the two-hybrid system. Boi2p has a proline-rich sequence that is critical for displaying the Boi2p-Bem1p two-hybrid interaction, an SH3 domain in its NH2-terminal half, and a pleckstrin homology domain in its COOH-terminal half. A BOI2 homologue, BOI1, was identified as a gene whose overexpression inhibited cell growth. Cells overexpressing either BOI1 or BOI2 were arrested as large, round, and unbudded cells, indicating that the Boi proteins affect cell polarization. Genetic analysis revealed that BOI1 and BOI2 are functionally redundant and important for cell growth. delta boi1 delta boi2 cells became large round cells or lysed with buds, displaying defects in bud formation and in the maintenance of cell polarity. Analysis using several truncated versions of BOI2 revealed that the COOH-terminal half, which contains the pleckstrin homology domain is essential for the function of Boi2p in cell growth, while the NH2- terminal half is not, and the NH2-terminal half might be required for modulating the function of Bem1p. Overproduction of either Rho3p or the Rho3p-related GTPase Rho4p suppressed the boi defect. These results demonstrate that Rho3p GTPases and Boi proteins function in the maintenance of cell polarity for bud formation.     

Modifications of substituted seryl and threonyl residues in phosphopeptides and a polysialoglycoprotein by beta-elimination and nucleophile additions

The beta-elimination and nucleophile addition reactions of the substituted serine and threonine residues were studied using several synthesized fluorescence-labeled phosphopeptides and a salmon egg polysialoglycoprotein (PSGP). The reagents used were 1 M CH3SH-0.43 M NaOH, 1 M NaBH4-0.1 M NaOH, 1 M CH3NH2-0.1 M NaOH, and 1 M Na2SO3-0.1 M NaOH. The beta-elimination reaction of a phosphoserine peptide, Gly-Ser(PO4)-Glu-AEAP, was about 20 times faster than that of the corresponding phosphothreonine peptide. The carboxyl-side amino acid of the phosphoamino acids in peptides greatly affected the beta-elimination rate. The beta-elimination reaction rates of O-glycosyl serine and threonine in the polysialoglycoprotein were similar and were about a half of that of the phosphoserine peptide. The rates of addition of the three nucleophiles and hydrogen to alpha-aminoacrylic acid (beta-elimination product of substituted serine) in the peptide decreased in the order of CH3SH, Na2SO3, CH3NH2, and H2(NaBH4), and the addition to alpha-aminocrotonic acid (beta-elimination product of substituted threonine) in the order of Na2SO3, CH3NH2, CH3SH, and H2. These results indicated that sulfite is the most recommended nucleophile because of its high addition rate. If sulfite addition is carried out in the presence of NaBH4, sugar chains can be released as alditols, converting the sugar-attaching amino acids to beta-sulfoamino acids.     

Affinity labeling of the primary bilirubin binding site of human serum albumin.

A label for the bilirubin binding sites of human serum albumin was synthesized by reacting 2 mol of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) with 1 mol of bilirubin. This yielded a water-soluble derivative in which both carboxyl groups of bilirubin were converted to reactive enol esters. Covalent labeling was achieved by reacting the label with human serum albumin under nitrogen at pH 9.4 and 20 degrees. Under the same conditions, no covalent binding to the monomers of several proteins could be demonstrated. The number of binding sites for bilirubin and the label were found to be the same, and competition experiments with bilirubin showed inhibition of covalent labeling. The absorption, fluorescence and CD spectra of the label in a complex with human serum albumin were similar to those of the bilirubin human serum albumin complex. However, following covalent attachment to the spectral properties were changed, indicating loss of conformational freedom of the chromophore. Labeling ratios were selected to result in the incorporation of less than 1 mol of label/mol of human serum albumin. Under these conditions, labeling is thought to occur primarily at the high affinity binding site.     

Accessibilities of the sulfhydryl groups of native and photooxidized lens crystallins: a fluorescence lifetime and quenching study

Fluorescence lifetime and acrylamide quenching studies on the N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS)-labeled sulfhydryl groups of bovine lens alpha-, beta H-, and gamma-crystallins were carried out to characterize the microenvironment of the sulfhydryls and changes produced by singlet oxygen mediated photooxidation. For the untreated proteins, the lifetimes of the major decay component of the fluorescence-labeled crystallins were 15.2, 14.4, and 13.0 ns, and the quenching rate constant, kq, values were 16.6 x 10(7), 26.9 x 10(7), and 32.7 x 10(7) M-1 s-1 for alpha-, beta H-, and gamma-crystallins, respectively. The results indicate that as the polarity of the sulfhydryl site increased (i.e., its lifetime decreased), its accessibility to collisional quenching by acrylamide also increased. The minor decay component of the fluorescence label was not significantly quenched by acrylamide for all three classes of crystallins. When the proteins were irradiated in the presence of methylene blue, in a system generating singlet oxygen, the kq value for acrylamide quenching of the major decay component of alpha-crystallin decreased to zero, while its lifetime decreased to 6 ns. Neither the lifetime nor the kq of alpha-crystallin recovered completely in the presence of the singlet oxygen quencher sodium azide. Light-induced binding of the photosensitizer methylene blue to the crystallins was observed by absorption spectroscopy. The bound photosensitizer partially quenches the fluorescence lifetime of the N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) label in irradiated alpha-crystallin. Further decrease in the lifetime occurs as a result of the singlet oxygen mediated conformational change. The results suggest that the fluorescence lifetime of the AEDANS is fully quenched in the irradiated alpha-crystallin and there is no further quenching by acrylamide. An increase in the fraction of the minor component of beta H-crystallin which was inaccessible to acrylamide quenching was observed after irradiation. There was no effect of irradiation on the kq for acrylamide quenching of the major component of the decay of AEDANS bound to beta H- or gamma-crystallins. Static quenching was found to contribute significantly to the steady-state quenching plots of the polar sulfhydryl sites of irradiated alpha-crystallin and of untreated and irradiated beta H- and gamma-crystallins, but it had no detectable role in the case of untreated alpha-crystallin. Fluorescence anisotropy of the AEDANS label bound to the crystallins was higher in the irradiated crystallins as compared with the controls.     

The location of sulphydryl groups in alpha-crystallin

The microenvironments of the sulphydryl groups in the multimeric protein, alpha-crystallin, were studied by examining: the rate of the reaction of the groups with DTNB; the effect of increasing urea concentrations on their accessibilities; and the quenching of a fluorescent probe. In foetal bovine alpha-crystallin (1 SH/alpha A subunit) both kinetic and quenching studies indicated that over 90% of the sulphydryl groups fell into a single buried class; the remainder was exposed. In the human protein (2 SH/alpha A subunit), half of the groups were buried and the other half exposed. Accessible sulphydryl groups increased gradually as the urea concentration was increased, with complete exposure at about 4.0 M. Sedimentation velocity analyses revealed that no significant dissociation of the aggregates into subunits occurred below 3.5 M urea, at which point over 80% of the sulphydryl groups were exposed. An age-dependent increase (3-35%) was found in the proportion of exposed sulphydryl groups in bovine alpha-crystallin and a decrease in the urea concentration required to expose the remainder. It was concluded that the single cysteine is buried in the newly synthesized protein, but becomes solvent-exposed as a result of age-related conformational changes. Our observations are consistent with a quaternary structure in which all alpha A subunits occupy equivalent sites.     

Detection of local polarity and conformational changes at the active site of rabbit muscle creatine kinase with a new arginine-specific fluorescent probe

A new polarity-sensitive fluorescent probe, 3-(4-chloro-6-p-glyoxal-phenoxy-1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine (CGTDP), is synthesized for selective labeling of active-site arginine residues. The probe comprises a neutral red moiety as a polarity-sensitive fluorophore and a phenylglyoxal unit as an arginine-specific labeling group. The probe exhibits a sensitive response of shift of fluorescence maximum emission wavelength to solvent polarity only instead of pH or temperature, which leads to the use of the probe in detecting the local polarity and conformational changes of the active site of rabbit muscle creatine kinase (CK) denatured by pH or temperature. The polarity of the active site domain has been first found to correspond to a dielectric constant of about 44, and the conformational change of the active site directly revealed by CGTDP occurs far before that of CK as a whole disclosed by the intrinsic tryptophan fluorescence during acid or thermal denaturation. The present strategy may provide a useful method to detect the local polarity and conformational changes of the active sites of many enzymes that employ arginine residues as anion recognition sites under different denaturation conditions.     

Active site selective labeling of serine proteases with spectroscopic probes using thioester peptide chloromethyl ketones: demonstration of thrombin labeling using N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl

The feasibility of a new approach to incorporation of spectroscopic probes into the active sites of certain serine proteases has been demonstrated. The method is based on inactivation of a serine protease with a thioester derivative of a peptide chloromethyl ketone. The thiol group generated by reaction of the covalent enzyme-inhibitor complex with NH2OH provides a unique site for subsequent labeling with thiol-reactive probes. To evaluate the method, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl was synthesized by reaction of the thrombin-specific tripeptide chloromethyl ketone with succinimidyl (acetylthio)acetate and purified by sulfopropyl-Sephadex and Sephadex G-10 chromatography. Reverse-phase high-performance liquid chromatography indicated that the product was 90 +/- 3% pure. The compound was quantitated by using 5,5'-dithiobis(2-nitrobenzoic acid) to measure the concentration of thiol produced in the presence of NH2OH. On this basis, titrations of the irreversible loss of human alpha-thrombin activity had end points of 1.1 +/- 0.1 mol of inhibitor/mol of active sites, indicating a 1:1 stoichiometry for inactivation. Incubation of N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-thrombin with 5-(iodoacetamido)fluorescein in the presence of NH2OH resulted in incorporation of 0.96 mol of the fluorescence probe/mol of active sites and the appearance of fluorescein fluorescence associated with the active site containing B-chain on sodium dodecyl sulfate-polyacrylamide gels. Fluorescence labeling of thrombin required reaction of the inhibitor at the active site as well as subsequent generation of the thiol group with NH2OH. It is concluded that active site selective labeling can be achieved by using this approach, which is likely to be applicable to other proteases, peptide chloromethyl ketones, and a wide variety of probes.     

Differential behavior of the two free sulfhydryl groups of human plasma fibronectin: effects of environmental factors.

We report here a novel approach to label specifically one of the two cryptic, free sulfhydryl groups per subunit of human plasma fibronectin with either an 15N,2H-maleimide spin label or a coumarinylphenyl maleimide fluorescent label. This permits the use of electron spin resonance (ESR) or fluorescence techniques to study molecular dynamics of fibronectin with the label attached to a single site per chain on the protein molecule. The method is based on our observation that upon adsorption of fibronectin to a gelatin-coated surface, the SH1 site, located between the DNA-binding and the cell-binding domains, is partially exposed, while the SH2 site, located within the carboxyl-terminal fibrin-binding domain, remains buried and unreactive. The procedures for the preparation of the selectively labeled fibronectins are described in detail. The physicochemical properties of these single-site labeled fibronectins, particularly as affected by high salt, heparin, surface binding, and temperature, were characterized by ESR spin-label and steady-state fluorescence techniques. The steady-state fluorescence measurement indicates that both local environments of SH1 and SH2 sites are relatively hydrophobic, and that the SH2 site is more hydrophobic than the SH1 site. The ESR results show that heparin or high salt induces an increase in the domainal flexibility in both SH1 and SH2 regions, perhaps through the disruption of domain-domain interactions in the fibronectin molecule, and that the former is more effective than the latter in producing such an effect. The observed heparin effect is reversible by addition of calcium ions in the SH2 regions but not in the SH1 regions. In addition, at temperatures above 44 degrees C, both type III homologous regions containing the free sulfhydryl groups are shown to undergo denaturation and aggregation processes. The data presented here suggest that the newly developed method for differential labeling of the free sulfhydryl groups in fibronectin should be useful for mapping the spatial arrangement of structural domains in the protein molecule using spin-label-spin-probe and fluorescence energy transfer techniques.     

Immunolocalization of venom metalloproteases in venom glands of adult and of newborn snakes of Bothrops jararaca

Using immunoelectronmicroscopy we analyzed qualitative and quantitatively the intracellular distribution of bothropasin, hemorrhagic factor 2 (HF2) and hemorrhagic factor 3 (HF3) in the venom secretory cells from adult snakes in the active (7 days after venom extraction) and in the resting (without venom extraction for 40 days) stages of protein synthesis. Glands from the newborn Bothrops jararaca were also studied. The results lead to the conclusion that all the secretory cells and the secretory pathway in the cells are qualitatively alike in regard to their content of the three metalloproteases. Secretory cells from the resting glands, unlike the active ones and the newborn glands, did not present immunolabeling in the narrow intracisternal spaces of the rough endoplasmic reticulum (RER). The label intensity for bothropasin was greater than that for the other proteins in the adults. HF3 and HF2 labeling densities in the newborn were higher than in the adults and HF3 labeling was not different from that of bothropasin. Co-localization of the three metalloproteases was detected in the RER cisternae of the active gland secretory cells, implying that mixing of the proteases before co-packaging into secretory vesicles occurs at the beginning of protein synthesis in the RER cisternae.     

Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. II. Properties of thrombin derivatives as reporters of prothrombin fragment 2 binding and specificity of the labeling approach for other proteinases.

The behavior of an array of fluorescent human alpha-thrombin derivatives in reporting binding of the fragment 2 domain of prothrombin was characterized as a representative application of the active-site-selective labeling approach to studies of blood coagulation proteinase regulatory interactions. An array of 16 thrombin derivatives was prepared by affinity labeling of the proteinase active site with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl or N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, followed by selective modification of the NH2OH-generated thiol group on the covalently incorporated inhibitors with each of eight thiol-reactive fluorescence probes. The changes in probe fluorescence intensity of the derivatives, signaling changes in the environment of the catalytic site associated with fragment 2 binding, appeared to be a unique and unpredictable function of the structure of the probe and the connecting peptide. These results demonstrated the utility of the labeling approach for overcoming the problem of not being able to predict which fluorescent label will provide the most useful proteinase derivative for investigating an interaction by enabling a greater variety of them to be prepared and screened for those with the most desirable properties. To determine whether the approach could be extended to other proteinases, the specificity of labeling with the fluorescence probe iodoacetamide, 5-(iodoacetamido)fluorescein, by use of the two thioester inhibitors was evaluated for several other blood coagulation proteinases and related trypsin-like enzymes. All of the proteinases were labeled in an active-site-selective manner. The combined results of quantitating the labeling reactions for the proteinase and inhibitor combinations studied thus far showed active-site-specific incorporation of 0.98 +/- 0.10 mol of inhibitor/mol of active sites and 0.92 +/- 0.11 mol of probe/mol of active sites, representing an overall greater than or equal to 93% site-specificity of labeling. These results demonstrated the broad applicability of the labeling approach for fluorescence studies of proteinases that differ greatly in their catalytic specificities.     

Relocation of the disulfonic stilbene sites of AE1 (band 3) on the basis of fluorescence energy transfer measurements

Previous fluorescence resonance energy transfer (FRET) measurements, using BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) as a label for the disulfonic stilbene site and FM (fluorescein-5-maleimide) as a label for the cytoplasmic SH groups on band 3 (AE1), combined with data showing that the cytoplasmic SH groups lie about 40 A from the cytoplasmic surface of the lipid bilayer, would place the BIDS sites very near the membrane's inner surface, a location that seems to be inconsistent with current models of AE1 structure and mechanism. We reinvestigated the BIDS-FM distance, using laser single photon counting techniques as well as steady-state fluorescence of AE1, in its native membrane environment. Both techniques agree that there is very little energy transfer from BIDS to FM. The mean energy transfer (E), based on three-exponential fits to the fluorescence decay data, is 2.5 +/- 0.7% (SEM, N = 12). Steady-state fluorescence measurements also indicate <3% energy transfer from BIDS to FM. These data indicate that the BIDS sites are probably over 63 A from the cytoplasmic SH groups, placing them near the middle or the external half of the lipid bilayer. This relocation of the BIDS sites fits with other evidence that the disulfonic stilbene sites are located farther toward the external membrane surface than Glu-681, a residue near the inner membrane surface whose modification affects the pH dependence and anion selectivity of band 3. The involvement of two relatively distant parts of the AE1 protein in transport function suggests that the transport mechanism requires coordinated large-scale conformational changes in the band 3 protein.     

Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. I. Specificity of thrombin labeling.

In a new strategy for labeling the active sites of serine proteinases with fluorescence probes (Bock, P. E. (1988) Biochemistry 27, 6633-6639), a thioester peptide chloromethyl ketone inhibitor is incorporated into the enzyme active center and used to produce a unique thiol group which provides a site for selective chemical modification with any one of many thiol-reactive fluorescence probes. This approach was developed to increase the opportunities for identifying fluorescent proteinase derivatives that act as reporters of binding interactions by allowing a large number of derivatives, representing a broad range of probe spectral properties, to be readily prepared. In the studies described here, the specificity of the labeling approach was evaluated quantitatively for the labeling of human alpha and beta/gamma-thrombin with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl and N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, and the thiol-reactive fluorescence probe, 5-(iodoacetamido)fluorescein. Irreversible inactivation of thrombin by the inhibitors was accompanied by incorporation of 0.98 +/- 0.06 mol/mol of the thioester group into the active site, independent of a 470-fold difference between the thioester peptide chloromethyl ketones in the bimolecular rate constants of alpha-thrombin affinity labeling. Subsequent mild treatment of the covalent thrombin-inhibitor complexes with NH2OH in the presence of 5-(iodoacetamido)fluorescein resulted in generation of the thiol group together with its selective modification and incorporation of 0.96 +/- 0.07 mol of probe/mol of active sites. The incorporated label was localized to a 9000 molecular weight region of alpha and beta/gamma-thrombin containing the catalytic-site histidine residue. Evaluation of competing, side reactions showed that they did not significantly compromise the active site specificity of labeling. These results demonstrated equivalent, active-site-selective fluorescence probe labeling of alpha and beta/gamma-thrombin by use of either of the thioester peptide chloromethyl ketones, with a site specificity of greater than or equal to 94%.     

CLASI-FISH: Principles of combinatorial labeling and spectral imaging

Although the number of phylotypes present in a microbial community may number in the hundreds or more, until recently, fluorescence in situ hybridization has been used to label, at most, only a handful of different phylotypes in a single sample. We recently developed a technique, CLASI-FISH for combinatorial labeling and spectral imaging – fluorescence in situ hybridization, to greatly expand the number of distinguishable taxa in a single FISH experiment. The CLASI technique involves labeling microbes of interest with combinations of probes coupled with spectral imaging to allow the use of fluorophores with highly overlapping excitation and emission spectra. Here, we present the basic principles and theory of CLASI-FISH along with some guidelines for performing CLASI-FISH experiments. We further include a protocol for creating fluorescence spectral reference standards, a vital component of successful CLASI-FISH.     

Inhibitory effects and underlying mechanism of 7-hydroxyflavone phosphate ester in HeLa cells

Chrysin and its phosphate ester have previously been shown to inhibit cell proliferation and induce apoptosis in Hela cells; however, the underlying mechanism remains to be characterized. In the present study, we therefore synthesized diethyl flavon-7-yl phosphate (FP, C(19)H(19)O(6)P) by a simplified Atheron-Todd reaction, and explored its anti-tumor characteristics and mechanisms. Cell proliferation, cell cycle progression and apoptosis were measured by MTS, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling techniques, respectively in human cervical cancer HeLa cells treated with 7-hydroxyflavone (HF) and FP. p21, proliferating cell nuclear antigen (PCNA) and cAMP levels in Hela cells were analyzed by western blot and radioimmunoassay. Both HF and FP inhibited proliferation and induced apoptosis in HeLa cells via induction of PCNA/p21 expression, cleaved caspase-3/poly (ADP-ribose) polymerase (PARP)-1, elevation of cAMP levels, and cell cycle arrest with accumulation of cells in the G0/G1 fraction. The effects of FP were more potent than those of HF. The interactions of FP with Ca(2+)-calmodulin (CaM) and Ca(2+)-CaM-phosphodiesterase (PDE)1 were explored by electrospray ionization-mass spectrometry and fluorescence spectra. FP, but not HF, formed non-covalent complexes with Ca(2+)-CaM-PDE1, indicating that FP is an inhibitor of PDE1, and resulting in elevated cellular cAMP levels. It is possible that the elevated cAMP levels inhibit growth and induce apoptosis in Hela cells through induction of p21 and cleaved caspase-3/PARP-1 expression, and causing down-regulation of PCNA and cell cycle arrest with accumulation of cells in the G0/G1 and G2/M fractions. In conclusion, FP was shown to be a Ca(2+)-CaM-PDE inhibitor, which might account for its underlying anti-cancer mechanism in HeLa cells. These observations clearly demonstrate the special roles of phosphorylated flavonoids in biological processes, and suggest that FP might represent a potential new drug for the therapy of human cervical carcinoma.     

Reactive disulfides trigger Ca2+ release from sarcoplasmic reticulum via an oxidation reaction

Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to the S-S bond such as 2,2'-dithiodipyridine (2,2'-DTDP), 4,4'-dithiodipyridine, and N-succinimidyl 3(2-pyridyldithio)propionate (SPDP) trigger Ca2+ release from sarcoplasmic reticulum (SR) vesicles. They are known to specifically oxidize free SH sites via a thiol-disulfide exchange reaction with the stoichiometric production of thiopyridone. Thus, the formation of a mixed S-S bond between an accessible SH site on an SR protein and a RDS causes large increases in SR Ca2+ permeability. Reducing agents, glutathione (GSH) or dithiothreitol reverse the effect of RDSs and permit rapid re-uptake of Ca2+ by the Ca2+, Mg2+-ATPase. The RDSs, 2,2'-DTDP, 4,4'-dithiodipyridine and SPDP displaced [3H]ryanodine binding to the Ca2+-receptor complex at IC50 values of 7.5 +/- 0.2, 1.5 +/- 0.1, and 15.4 +/- 0.1 microM, respectively. RDSs did not alter the rapid initial phase of Ca2+ uptake by the pump, stimulated ATPase activity, and induced release from passively loaded vesicles with nonactivated pumps; thus they act at a Ca2+ release channel and not at the Ca2+, Mg2+-ATPase. Efflux rates increased in 0.25-1.0 mM [Mg2+]free then decreased in 2-5 mM [Mg2+]free. Adenine nucleotides inhibited the oxidation of SHs on SR protein by RDSs and thus reduced Ca2+ efflux rates. However, once RDSs oxidized these SH sites and opened the Ca2+ release pathway, subsequent additions of nucleotides stimulated Ca2+ efflux. In skinned fibers, 2,2'-dithiodipyridine elicited rapid twitches which were blocked by ruthenium red. These results indicate that RDSs trigger Ca2+ release from SR by oxidizing a critical SH group, and thus provide a method to covalently label the protein(s) involved in causing these changes in Ca2+ permeability.     

Modification of alpha-crystallin subunit chains and formation of alpha-neoprotein molecules: role of SH groups

Immunoadsorbents with bound antibodies restricted to determinants dependent on alpha-crystallin's quaternary structure permitted the fractionation of the population of 125I-labeled alpha-crystallin molecules, treated by iodoacetic acid, into molecules in which the native structure was still preserved and molecules with a completely different quaternary structure than the native protein. Parallel experiments with [14C]iodoacetic acid yielded information on the percentage of blocked SH groups in each of the above two fractions. The presence of molecules formed by A with B-chain association was established by sequential binding first to an immunoadsorbent with antibodies restricted to determinants located on alpha-crystallin's A-subunit chains as ligand and second, after desorption, to an immunoadsorbent with antibodies to B chains as ligand. With the aid of these techniques, it was established that (i) The modified alpha-crystallin molecules with quaternary determinants of the native protein contained a maximum of 23% blocked SH groups, indicating that the carboxymethylation involved only the fast-reacting surface SH groups. (ii) The modified alpha-crystallin molecules without the native protein's quaternary structure were built by a different association between A and B subunits than in alpha-crystallin, indicating formation of alpha-neoprotein molecules. (iii) Monomeric A chains with all SH groups carboxymethylated, and monomeric B chains in a ratio of 1A:5B, 2A:1B, and 5A:1B in urea solution, associate on dialysis, forming alpha-neoprotein molecules.