The conversion of azo-quenchers to fluorophores.

In this short note we describe the conversion of the widely used fluorescence quenching azo-dyes DABCYL and HABA to fluorophores. The dyes were conjugated to the proteins RNase and human serum albumin (HSA) and subsequently reduced using sodium dithionite (Na2S2O4), thus forming amine-containing fluorophores. Since this chemical reaction can be applied to any azo-containing quencher compound, a great variety of substances can be readily obtained synthetically. This approach provides a promising tool in the use of fluorescence-based investigations of biomolecular interactions.

Fast mass spectrometry detection of tryptophan-containing peptides and proteins by reduction with pyridine-borane.

In this note, we describe a method devised to detect, by means of mass spectrometry (MS), tryptophan-containing peptides and proteins using pyridine-borane. This reagent selectively reduces tryptophan residues, converting them to 2,3-dihydro-tryptophan, thereby enabling quantitation of tryptophans.

Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes.

Fluorescent derivatives of gangliosides were prepared by oxidizing the sialyl residues to aldehydes and reacting them with fluorescent hydrazides. When rhodaminyl gangliosides were incubated with lymphocytes, the cells incorporated them in a time- and temperature-dependent manner. Initially, the gangliosides were evenly distributed on the cell surface but were redistributed into patches and caps by antirhodamine antibodies. When the cells were then stained with a second antibody or protein A labeled with fluorescein, the fluorescein stain revealed the coincident movement of both the gangliosides and the antirhodamine antibodies. When the cells were treated with both rhodamine and Lucifer yellow CH-labeled gangliosides, the antirhodamine antibodies induced patching and capping of both fluorescent gangliosides but had no effect on cells incubated only with Lucifer yellow CH-labeled gangliosides. In addition, capping was observed on cells exposed to cholera toxin, antitoxin antibodies, and rhodamine-labeled protein A, indirectly showing the redistribution of endogenous ganglioside GM1, the cholera toxin receptor. By incorporating Lucifer yellow CH-labeled GM1 into the cells and inducing capping as above, we were able to demonstrate directly the coordinate redistribution of the fluorescent GM1 and the toxin. When the lymphocytes were stained first with Lucifer yellow CH-labeled exogenous ganglioside GM3, which is not a toxin receptor, there was co-capping of endogenous GM1 (rhodamine) and exogenous GM3 (Lucifer yellow CH). These results suggest that gangliosides may self-associate in the plasma membrane which may explain the basis for ganglioside redistribution and capping.

A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein.

The 25-kD inhibitor of actin polymerization (25-kD IAP), isolated from turkey smooth muscle (Miron, T., M. Wilchek, and B. Geiger, 1988. Eur. J. Biochem. 178:543-553), is shown here to be a low molecular mass heat shock protein (HSP). Direct sequence analysis of the purified protein, as well as cloning and sequencing of the respective cDNA, disclosed a high degree of homology (67% identity, 80% similarity) to the human 27-kD HSP. Southern blot of chicken genomic DNA disclosed one band, suggesting the presence of a single gene, and Northern blot analysis revealed abundant transcript of approximately 1 kb in gizzard and heart tissues and lower amounts in total 18-d chick embryo RNA and in cultured fibroblasts. Exposure of the latter cells to 45 degrees C resulted in over 15-fold increase in the apparent level of the 25-kD IAP protein, confirming that its expression is regulated by heat shock. Immunofluorescent microscopic localization indicated that after heat treatment, the levels of the 25-kD IAP were markedly increased and the protein was apparently associated with cytoplasmic granules. Heat shock also had a transient, yet prominent, effect on the microfilament system in cultured fibroblasts: stress fibers disintegrated within 10-15 min after incubation at 45 degrees C, yet upon further incubation at the elevated temperature, conspicuous actin bundles were apparently reformed.

Staphylococcal nuclease: size and specificity of the active site.

The dissociation constants and standard free energies of complex formation determined with staphylococcal nuclease and a series of 5'-phosphoryloligothymidyl derivatives of increasing chain length suggest that maximum stability is reached with an oligonucleotide containing three nucleotide units. A proposed model of the active site that contains other knowledge of the specificity and the catalytic mechanism of this enzyme postulates the existence of three nonequivalent phosphate binding subsites and a closely related phosphodiester hydrolytic subsite.

Avidin-biotin technology ten years on: has it lived up to its expectations?

Following the practical success attributed to avidin-biotin technology, researchers are now returning to the basics to try to understand the structural and functional requirements for the high-affinity avidin-biotin interaction.

Affinity purification of the avidin protein family, based on crystal structures of avidin-HABA complexes.

Since the importance of the high affinity between avidin and biotin, Kd = 3 × 10-16 M, gained universal recognition, numerous chemical, biological and medical avidin-biotin based applications have been developed. However, in some cases the high affinity may be a disadvantage, as this interaction is irreversible under physiological conditions. The dye, 4'-hydroxyazobenzene-2-carboxylic acid (HABA), binds avidin, at the biotin binding site, as determined by X-ray, at a much lower affinity constant, Kd = 6 × 10-6 M. We prepared a HABA affinity column (amber colored). Avidin bound to the column at a pH between 4 and 8.5, causing a change of color to red, and it could be eluted at mild conditions with buffers containing biotin, HABA, 1.5 M potassium chloride or a pH lower than 4.0 or higher than 8.5. Avidin eluted with HABA, created a red avidin-HABA complex, which was visualized. HABA free avidin was obtained by dialysis, which was followed by the loss of red coloration. The novel and easy to use HABA-affinity column was employed in our lab to prepare pure, fully glycosylated avidin from egg white. Most importantly, it may serve as an ideal tool for educational purposes, illuminating concepts of molecular recognition, reversible molecular binding, structure-based molecular design and solid phase chemical synthesis, as it is a reliable and visible reagent.

Chiral discrimination using an immunosensor.

Based on the stereoselectivity of immunoglobulins, we have developed a new chiral sensor for the detection of low-molecular-weight analytes. Using surface plasmon resonance detection, enantiomers of free, underivatized alpha-amino acids can be monitored in a competitive assay by their interaction with antibodies specific for the chiral center of this class of substances. The sensitivity to the minor enantiomer in nonracemic mixtures exceeds currently available methods; therefore, such immunosensors can readily detect traces of enantiomeric impurities and are attractive for a range of applications in science and industry.

Higher antitumor efficacy of daunomycin when linked to dextran: in vivo and in vitro studies.

Daunomycin was coupled to dextrans of various molecular sizes. The binding to the dextran carriers augmented the therapeutic efficacy of the antitumor agent in a murine lymphoma line (YAC). When the treatment with the drug or its conjugates was given concomitantly with the tumor cells at separate sites, the unbound drug was able, at its optimally effective doses, to prevent tumors in 40% of the mice, whereas the drug-dextran was efficient in 80% of the mice. The advantage of the drug-dextran over the free drug was also manifested when the treatment was given 6 days after tumor transplantation. However, a further delay of the treatment resulted in a decrease in the potency of the drug-dextran. Similar behavior was observed when increasing tumor loads were transplanted (10(5)-10(8) cells) and when the treatment was administered immediately. The most favorable effect of the drug-dextran was obtained with 10(7) cells, but against 10(8) cells neither the free drug nor the bound one was effective.

Cytotoxicity of streptavidin-blocked biotinyl-ricin is retrieved by in vitro immunotargeting via biotinyl monoclonal antibody.

The streptavidin-biotin system has been used to immunotarget whole ricin to tumor cells in a system that overcomes ricin-nonspecific cytotoxicity. Biotin was linked to ricin via a disulfide-containing reagent, sulfosuccinimidyl-2-(biotinamido)ethyl-1,3'-dithiopropionate. The product, biotinyl-S,S-ricin (b-ricin), retained most of its in vitro cytotoxic activity against human epidermoid carcinoma (KB) cells. Complexing b-ricin to streptavidin resulted in greater than 99% loss of its cellular toxicity which is associated with loss of cell-binding activity. The streptavidin-b-ricin complex could, however, be targeted to KB cells via the biotinylated monoclonal antibody 108 which is specific to the epidermal growth factor receptor overexpressed on KB cells. The complex did not regain its activity if the specific antibody was not biotinylated or if the biotinylated antibody was of a different specificity. Streptavidin is thus used to block b-ricin, presumably due to a steric restraint of the streptavidin on the ricin B-chain, and to bridge it to biotinyl antibody recognizing the target cell. Avidin could not replace streptavidin in this system since a complex between b-ricin and avidin retained a major part (60%) of ricin cytotoxic activity. This is attributed to the nonspecific binding of avidin to cells in vitro, including the KB cells. It is suggested that b-ricin is blocked by both streptavidin and avidin, but once the complex gains access to the cell surface, its cytotoxic activity is specifically retrieved.

The covalent binding of daunomycin and adriamycin to antibodies, with retention of both drug and antibody activities.

Daunomycin and adriamycin, two potent cancer chemotherapeutic agents, were linked to immunoglobulins, making use of various covalent cross-linking methods. The most suitable method for binding of the drugs to the antibodies, which retained both antibody and drug activity, was periodate oxidation of the drug, followed by the linking of the oxidized drug to the immunoglobulin and subsequent reduction of the product with sodium borohydride. The activity of the drug-antibody conjugates was tested in vitro on tumor and normal cell cultures and was found to be similar to that of the free drug. A significant amount of antibody activity was retained, as found both with anti-bovine serum albumin antibodies, assayed by chemically modified bacteriophage, and with anti-mouse tumor antibodies, assayed by C'-dependent cytotoxicity.

Modification of a specific tyrosine residue of ribonuclease A with a diazonium inhibitor analog.

The diazonium salt of 5'-(4-aminophenyl phosphoryl)-uridine 2'(3')-phosphate reacts stoichiometrically with pancreatic ribonuclease and modifies only one tyrosyl residue, which was identified as Tyr 73 in the amino acid sequence. The modification does not inhibit the biological activity of RNAase on ribonucleic acid, although a large change in the binding constant towards cytidine cyclic phosphate was observed. The modification can be inhibited by addition of the competitive inhibitor cytidine 2'(3')5'-diphosphate and may indicate that Tyr 73 is the most exposed residue or has a unique reactivity towards this reactive substrate analog.

Modulation of the catalytic properties of alpha-chymotrypsin by chemical modification at Tyr 146.

alpha--Chymotrypsin was modified with three different diazonium salts derived from D--phenylalanine. All three reagents react selectively with tyrosine 146 on the surface of the enzyme. The spectral and enzymatic properties of the azochymotrypsins are characteristic for each of the proteins. Dependent on the structure of the reagent employed for modification, the activity towards rho-nitroanilide substrate can be higher or about the same as that of the native enzyme.

Reversibility of the affinity labelled-biotin transport system in yeast cells.

Transport of biotin by Saccharomyces cerevisiae is inhibited by biotynyl p-nitrophenyl ester. Conversion of the inhibited cells to spheroplasts or simple treatment with thiols results in a total restoration of vitamin transport. Biotynyl p-nitrophenyl ester-induced inhibition is not due to an intracellular accumulation of the vitamin and consequent regulation, but appears to be due to specific labelling of the transport system.

The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins.

Allicin (thio-2-propene-1-sulfinic acid S-allyl ester) is the main biologically active component of garlic clove extracts. Its biological activity was attributed to either antioxidant activity or thiol disulfide exchange. Antioxidant properties of both allicin and its precursor, alliin (+S-allyl-L-cysteine sulfoxide), were investigated in the Fenton oxygen-radical generating system [H2O2-Fe(II)]. Using the spin trapping technique and ESR, it was found that both compounds possessed significant antioxidant activity. The reaction between allicin and L-cysteine was studied by 1H and 13C-NMR, and a S-thiolation product, S-allylmercaptocysteine, was identified. Allicin irreversibly inhibited SH-protease papain, NADP(+)-dependent alcohol dehydrogenase from Thermoanaerobium brockii (TBAD), and the NAD(+)-dependent alcohol dehydrogenase from horse liver (HLAD). All the three enzymes could be reactivated with thiol containing compounds. Papain could be reactivated with glutathione, TBAD with dithiothreitol or 2-mercaptoethanol (2-ME) but not by glutathione, while HLAD could be reactivated only with 2-ME. This study demonstrates that in addition to its antioxidant activity, the major biological effect of allicin should be attributed to its rapid reaction with thiol containing proteins.

Close similarity among streptavidin-like, biotin-binding proteins from Streptomyces.

Two strains of Streptomyces venezuelae were found to produce high-affinity, biotin-binding proteins, termed streptavidin v1 and v2, respectively. Both proteins were isolated to purity, and their corresponding genes were cloned and sequenced. Compared to streptavidin from S. avidinii, streptavidin v1 had only a single amino acid substitution and streptavidin v2 showed 9 such differences. The substitutions were remarkably conservative, none of which affected the amino acid residues known to be important to the biotin-binding properties or to the structure of the tetrameric protein. The results also indicate that the biosynthesis of such biotin-binding proteins is not simply a curious anomaly in a single species of Streptomyces. It is suggested that the classification of S. avidinii as a unique species should be reconsidered. The occurrence of these proteins appears to be linked to the production of an unusual synergistic antibiotic complex.

The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity.

Allicin (diallyl thiosulfinate) is the main biologically active component of the freshly crushed garlic extracts. In the present work the ability of allicin to cross through membranes (artificial and biological) was studied. Partition coefficients of allicin in water/octanol, water/hexadecane and water/phospholipids mixtures were determined. Using phospholipid vesicles loaded with hydrophilic thiols (reduced glutathione or 2-nitro-5-thiobenzoate), we observed that allicin freely permeates through phospholipid bilayers and interacts with the SH groups. The reaction rate of allicin with SH containing molecules after crossing the membrane was the same as in solution. Fast diffusion and permeation of allicin across human red blood cell membranes was also demonstrated. Allicin does not induce leakage, fusion or aggregation of membrane. The high permeability of allicin through membranes may greatly enhance the intracellular interaction with thiols.

A novel approach for the topographical localization of glycolipids on the cell surface.

In this study we have developed a prototype system for distinguishing between the topographical distribution of glycolipids versus glycoproteins on the ultrastructural level. Direct modification of membrane-based sialic acids with biotin groups labels both glycolipids and glycoproteins. In this case, subsequent ultrastructural localization of biotinylated sites would not discern between these two classes of glycoconjugate in an unambiguous manner. When biotinylated cells are fixed prior to interaction with ferritin-conjugated avidin, the mean distance of marker molecules from the membrane bilayer is 8.0 nm. In contrast, if the cells are allowed to cap through the action of ferritin-avidin conjugates on unfixed cells, the average distance (13.0 nm) of the marker molecules appears even more distant from the membrane on the capped portion of the cell (uropod), whereas those on the head region are positioned in close proximity to the bilayer (3.7 nm). In order to exclusively label cell surface glycolipids on the ultrastructural level, bovine brain gangliosides were biotinylated in vitro and the haptenized gangliosides were incorporated into intact cells. In this case, marker molecules denoting the incorporated gangliosides were found in relatively close juxtaposition to the membrane surface, in a manner strikingly similar to the labeling pattern of the head region on capped cells. These results support the concept that, in the native state, the carbohydrate portion of glycolipids is positioned closer to the membrane bilayer than that of glycoproteins.

Mode of transport and possible mechanism of action of L-phenylalanine benzyl ester as an anti-sickling agent.

L-Phenylalanine benzyl ester (Phe-Bz) and a number of ester analogues prevent sickling of erythrocytes from sickle cell disease patients. The compounds tested exhibit anti-sickling activity in the concentration range 0.5-3.0 mM. A general feature of these compounds is the presence of two aromatic rings in their molecular structure. The anti-sickling agents rapidly enter the erythrocyte and are hydrolysed to their component molecules. Incubation of human erythrocytes with 3.0 mM L-phenylalanine for 30 min at 37 degrees C results in accumulation of 2.0 mmol L-phenylanine/l cells, while incubation of erythrocytes with 3.0 mM Phe-Bz under similar conditions results in the production of 4.0 mmol L-phenylalanine/l cells and an equivalent amount of benzyl alcohol. Both L-phenylalanine and benzyl alcohol are inhibitors of the gelation of deoxyhaemoglobin S (deoxy-HbS) in vitro. Moreover, Phe-Bz and related anti-sickling agents fluidize the lipid bilayer of the erythrocyte membrane, inhibiting several transport systems, including those for L-phenylalanine, uridine and sulphate ions, as well as the Na+ pump and the Na+/K+ cotransporter, but increasing the passive influx and efflux of both cations and anions. The accumulation of Phe-Bz hydrolysis products within the erythrocyte together with the effects of Phe-Bz on cation permeability result in the influx of water causing the cell to swell. Thus, treatment of erythrocytes with 3.0 mM Phe-Bz at 37 degrees C for 30 min causes an increase in mean cell volume of 14.8%, decreasing the mean intracellular haemoglobin concentration from 34 to 29.6 g%. The increase in cell volume caused by Phe-Bz and its analogues together with the direct effects of their hydrolysis products on HbS probably act in concert to bring about the anti-sickling effect.

S-Allylmercaptoglutathione: the reaction product of allicin with glutathione possesses SH-modifying and antioxidant properties.

The reaction between allicin (diallylthiosulfinate), the active component of garlic and reduced glutathione was investigated. The product of this reaction, mixed disulfide S-allylmercaptoglutathione (GSSA) was separated by high performance liquid chromatography and identified by 1H and (13)C nuclear magnetic resonance and mass spectroscopy. The reaction is fast (with an apparent bimolecular reaction rate constant of 3.0 M(-1) s(-1)). It is pH-dependent, which reveals a direct correlation to the actual concentration of mercaptide ion (GS(-)). Both GSSA and S-allylmercaptocysteine (prepared from allicin and cysteine) reacted with SH-containing enzymes, papain and alcohol dehydrogenase from Thermoanaerobium brockii yielding the corresponding S-allylmercapto proteins, and caused inactivation of the enzymes. The activity was restored with dithiothreitol or 2-mercaptoethanol. In addition, GSSA also exhibited high antioxidant properties. It showed significant inhibition of the reaction between OH radicals and the spin trap 5,5'-dimethyl-1-pyroline N-oxide in the Fenton system as well as in the UV photolysis of H2O2. In ex vivo experiments done with fetal brain slices under iron-induced oxidative stress, GSSA significantly lowered the production levels of lipid peroxides. The similar activity of GSSA and allicin as SH-modifiers and antioxidants suggests that the thioallyl moiety has a key role in the biological activity of allicin and its derivatives.