2-O-[2-(4,4'-dimethoxytrityloxyethyl)]-hydroxy acetaldehyde: a universal reagent for spectrophotometric estimation of polymer-supported functional groups

A new universal reagent, 2-O-[2-(4,4′-dimethoxytrityloxyethyl)]-hydroxy acetaldehyde (DEA), has been synthesized and used for the estimation of surface-bound aminoalkyl, aminooxyalkyl, hydrazinyl, and semicarbazide functions. The reaction completes in just 10 min in the case of aminoalkylated supports and 30 min in hydrazinyl supports, whereas it takes approximately 60 min in both aminooxyalkylated and semicarbazide-modified polymer supports. DEA-treated supports, including glass slides and PP films on exposure to acid, liberates 4,4′-dimethoxytrityl cation, which was measured spectrophotometrically to estimate these functionalities. The method estimates accessible functional groups, useful for calculating the quantity of the ligands to be immobilized.     

Conformational change of band 3 protein induced by diethyl pyrocarbonate modification in human erythrocyte ghosts

Diethyl pyrocarbonate inhibited the phosphate exchange across the human erythrocyte membrane. The exchange rate was inhibited only when the membranes were modified with the reagent from the cytosolic surface of resealed ghosts. The intracellular modification by diethyl pyrocarbonate inhibited the extracellular binding of [3H]dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid to band 3 protein. Furthermore, the extracellular 4,4'-dinitrostilbene-2,2'-disulfonic acid protected the membranes from the intracellular modification by diethyl pyrocarbonate. These results suggest that the extracellular binding of 4,4'-dinitrostilbene-2,2'-disulfonic acid to band 3 protein induces the conformational change of the intracellular counterpart of band 3 protein and the diethyl pyrocarbonate susceptible residue(s) is (are) hidden from the cytosolic surface of the cell membrane in connection with the conformational change. Conversely, under the conditions where the diethyl pyrocarbonate modification is confined to the intracellular side of the membrane, the extracellular binding site of [3H]dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid is hidden from the cell surface.     

A new synthetic protocol for labeled oligonucleotides, using a chemically cleavable universal linker

A two-step general method for labeling of synthetic oligonucleotides is described. The protocol employs a cleavable universal linker, 5'-O-(4,4'-dimethoxytrityl)-3'-O-benzoyl-2'-O-(2-cyanoethyl-N,N-diisopropyl)-uridine phosphoramidite, to effect coupling to polymer-bound oligonucleotide chains. Sequentially, coupling with commercially available phosphoramidite reagent of an appropriate label (Biotin, HEX etc.) in an automated DNA synthesizer is carried out. The labeled oligomers, obtained after cleavage and deprotection reactions, are analyzed on RP-HPLC. A distinctive feature of this protocol is the recovery of free oligomers from their labeled analogs under mild conditions. The oligomers obtained are comparable to the corresponding standard oligonucleotides (HPLC).     

Construction of oligonucleotide arrays on a glass surface using a heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA)

A rapid method for construction of oligonucleotide arrays on a glass surface, using a novel heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA), has been described. The heterobifunctional reagent, NTMTA, carries two different thermoreactive groups. The triethoxysilyl group on one end is specific towards silanol functions on the virgin glass surface, while the trifluoroethanesulfonyl (tresyl) group on the other end of the reagent reacts specifically with aminoalkyl- or mercaptoalkyl- functionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5′- aminoalkyl- or mercaptoalkyl-functionalized oligonucleotides were allowed to react with NTMTA to form a oligonucleotide-triethoxysilyl conjugate which, in a subsequent reaction with unmodified (virgin) glass microslide, results in surface-bound oligonucleotides. In the second route (B), the NTMTA reagent reacts first with a glass microslide whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with 5′-aminoalkyl or mercaptoalkyl oligonucleotides to generate support-bound oligonucleotides. Subsequently, the oligonucleotide arrays prepared by both routes were analyzed by hybridization experiments with complementary oligonucleotides. The constructed microarrays were successfully used in single and multiple nucleotide mismatch detection by hybridizing these with fluorescein-labeled complementary oligonucleotides. Further more, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides.     

A spectrophotometric method for the estimation of polymer-supported sulfhydryl groups

A sensitive and simple method is described for the quantitative determination of free sulfhydryl (-SH) groups on polymer supports. The method includes the reaction of 4,4'-dimethoxytrityloxy-S-(2-thio-5-nitropyridyl)-2-mercapto ethane (DTNPME) with polymer-supported sulfhydryl groups. After removal of excess reagent through washing, a weighed quantity of the polymer support is treated with perchloric acid to release the 4,4'-dimethoxytrityl cation from the polymer support into the solution. The dimethoxytrityl cation (lambda max = 498 nm, epsilon 498 = 70,000/M) is then quantified spectrophotometrically. A comparative study of the reagent DTNPME with 2,2'-dithiobis(5-nitropyridine) is also described.     

A spectrophotometric method for the estimation of amino groups on polymer supports

A simple and sensitive method for the estimation of polymer-supported amino groups is reported. The polymer support is treated either with N-succinimidyl-4-O-(4,4'-dimethoxytrityl)-butyrate or 2,4-dinitrophenyl-4-O-(4,4'-dimethoxytrityl)-butyrate and a catalytic amount of 4-dimethylaminopyridine. After removal of the excess reagent through washing, a weighed quantity of the polymer support is treated with perchloric acid to release the 4,4'-dimethoxytrityl cation from the solid support into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption (epsilon 498 = 70,000/M) at 498 nm, is determined spectrophotometrically. A comparative study of these reagents with N-succinimidyl-3-(2-pyridyldithio)-propionate, 4,4'-dimethoxytrityl chloride, and sodium 2,4,6-trinitrobenzenesulfonate methods is also included.     

Histochemical evidence for the differential surface labeling, uptake, and intracellular transport of a colloidal gold-labeled insulin complex by normal human blood cells

A colloidal gold-labeled insulin-bovine serum albumin (GIA) reagent has been developed for the ultrastructural visualization of insulin binding sites on the cell surface and for tracing the pathway of intracellular insulin translocation. When applied to normal human blood cells, it was demonstrated by both visual inspection and quantitative analysis that the extent of surface labeling, as well as the rate and degree of internalization of the insulin complex, was directly related to cell type. Further, the pathway of insulin (GIA) transport via round vesicles and by tubulo-vesicles and saccules and its subsequent fate in the hemic cells was also related to cell variety. Monocytes followed by neutrophils bound the greatest amount of labeled insulin. The majority of lymphocytes bound and internalized little GIA, however, between 5-10% of the lymphocytes were found to bind considerable quantities of GIA. Erythrocytes rarely bound the labeled insulin complex, while platelets were noted to sequester large quantities of the GIA within their extracellular canalicular system. GIA uptake by the various types of leukocytic cells appeared to occur primarily by micropinocytosis and by the direct opening of cytoplasmic tubulo-vesicles and saccules onto the cell surface in regions directly underlying surface-bound GIA. Control procedures, viz., competitive inhibition of GIA labeling using an excess of unlabeled insulin in the incubation medium, preincubation of the GIA reagent with an antibody directed toward porcine insulin, and the incorporation of 125I-insulin into the GIA reagent, indicated the specificity and selectivity of the GIA histochemical procedure for the localization of insulin binding sites.     

The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and 4,4'-dithiodipyridine.

Citrate synthase of Escherichia coli reacts rapidly with 1 equivalent of Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH. When the enzyme is treated instead with 4,4'-dithiodipyridine (4,4'-PDS), all activity is lost. Certain evidence in this paper is consistent with the belief that the sulfhydryl group modified by DTNB, and that whose modification by 4,4'-PDS inactivates the enzyme, are the same. (i) Both reagents abolish NADH fluorescence enhancement by the enzyme. (ii) Saturating levels of NADH and some other adenylic acid derivatives inhibit the reactions with both reagents. (iii) When the enzyme is modified with one equivalent of DTNB or 4,4'-PDS, subsequent reactivity toward the other reagent is greatly decreased. (iv) Following modifications, the DTNB and 4,4'-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a second enzyme sulfhydryl group, so that an enzyme disulfide is introduced. The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions. Certain evidence has also been found, however, that the sites of modification by DTNB and 4,4'-PDS are not the same. (i) DTNB modification desensitizes to NADH but does not inactivate, while 4,4'-PDS inactivates at least 99.9%. (ii) The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4'-PDS modified product. (iii) Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4'-PDS. The studies therefore indicate a close relationship between the DTNB desensitization and 4,4'-PDS inactivation, but they are unable to identify it exactly. Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB.     

Constraints imposed by protease accessibility on the trans-membrane and surface topography of the colicin E1 ion channel.

The surface topography of a 190-residue COOH-terminal colicin E1 channel peptide (NH2-Met 333-Ile 522-COOH) bound to uniformly sized 0.2-micron liposomes was probed by accessibility of the peptide to proteases in order (1) to determine whether the channel structure contains trans-membrane segments in addition to the four alpha-helices previously identified and (2) to discriminate between different topographical possibilities for the surface-bound state. An unfolded surface-bound state is indicated by increased trypsin susceptibility of the bound peptide relative to that of the peptide in aqueous solution. The peptide is bound tightly to the membrane surface with Kd < 10(-7) M. The NH2-terminal 50 residues of the membrane-bound peptide are unbound or loosely bound as indicated by their accessibility to proteases, in contrast with the COOH-terminal 140 residues, which are almost protease inaccessible. The general protease accessibility of the NH2-terminal segment Ala 336-Lys 382 excludes any model for the closed channel state that would include trans-membrane helices on the NH2-terminal side of Lys 382. Lys 381-Lys 382 is a major site for protease cleavage of the surface-bound channel peptide. A site for proteinase K cleavage just upstream of the amphiphilic gating hairpin (K420-K461) implies the presence of a surface-exposed segment in this region. These protease accessibility data indicate that it is unlikely that there are any alpha-helices on the NH2-terminal side of the gating hairpin K420-K461 that are inserted into the membrane in the absence of a membrane potential. A model for the topography of an unfolded monomeric surface-bound intermediate of the colicin channel domain, including a trans-membrane hydrophobic helical hairpin and two or three long surface-bound helices, is proposed.     

A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups

A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Biochem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (epsilon 498 = 70,000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4,4-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.     

Improved protein detection on an AC electrokinetic quartz crystal microbalance (EKQCM)

Microscale electrodes supplied with an AC field can generate rotational fluid patterns known as AC electroosmosis. In the present study, this effect was used to improve antibody binding on a biosensor surface. Antibodies, like many other large, slow moving biomolecules, tend to suffer from transport limitations during a reaction with a surface-bound receptor. Stirring such reactions with AC electroosmosis can alleviate this transport limitation by bringing fresh reagent to the surface. For the first time, the use of this phenomenon was used to improve the capture of protein on a sensor. Directly adsorbed antibodies were bound to the surface of specially modified quartz crystal microbalances, known as electrokinetic QCMs (EKQCMs) and the signal was enhanced by about 5.6 times. Modification of the QCM resulted in little reduction of quality factor (from ～ 5.3 k to ～ 4.6k) and an increased sensitivity to viscosity changes (151%). Full immunoassays performed on electrodes fabricated on glass surfaces were used to ensure antibody function was not significantly degraded by the enhancement technique.     

Studies of peptide antibiotics. XLIII. Syntheses of gramicidin S analogs containing D-serine or dehydroalanine in place of D-phenylalanine and asymmetric hydrogenation of the dehydroalanine residue

Gramicidin S (GS) analogs, [D-Ser4,4']-GS and its precursor [O-benzyl-D-Ser4,4']-GS, were synthesized by the conventional method in order to evaluate the role of the hydroxymethyl side chains in D-Ser at 4,4' positions on the biological activity. Another analog [L-Orn(delta-Boc)2,2',delta Ala4,D-Ser4']-GS was prepared from [D-Ser4,4']-GS by t-butyloxycarbonylation and successive dehydration using dicyclohexylcarbodiimide-CuCl as dehydrating reagent. The delta Ala residue was asymmetrically hydrogenated to D-Ala in the presence of Pd-black. On the microbial assays, [O-benzyl-D-Ser4,4']-GS showed high antimicrobial activity as natural GS, but [D-Ser4,4']-GS showed low activity; the structure-activity relationships of the analogs were discussed.     

Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein

A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.     

Evanescent resonator chips: a universal platform with superior sensitivity for fluorescence-based microarrays

In the present paper, we introduce for the first time a novel generation of a universal fluorescence transducer, the so-called evanescent resonator (ER) platform. The device comprises a transparent substrate and a thin dielectric surface layer containing sub-micron corrugated structures. The ER chip exhibits an inherent physical signal amplification due to confinement of excitation energy in the thin surface layer. Energy confinement is based on interference effects created by the abnormal reflection geometry and leads to efficient excitation of surface-bound fluorophores in the evanescent field of the chip. The evanescent resonator platform has the potential to increase the fluorescence yield of labelled biomolecules to more than 100-fold when compared with conventional microarray chips. The new ER device has been developed for analysis of nucleic acids from different species. However, it can be used with all kinds of biomolecular affinity systems. The platform combines superior sensitivity with exceptional reproducibility and ease of use. The chips are compatible with commercially available laser scanners, confocal microscopes, and portable or miniaturised CCD read-out equipment.     

Slow internalization of human chorionic gonadotropin by cultured granulosa cells

Kinetic studies were performed on two-day cultures of rat ovarian granulosa cells to follow the fate of surface-bound 125I-labeled human chorionic gonadotropin (125I-hCG). Low pH was used to release hCG from its surface receptor, allowing us to distinguish between surface-bound and internalized hormone. Because our results indicated that hormone is lost from the cell surface by dissociation as well as internalization, equations were derived to determine independent rate constants for each process. We calculate that if hormone binding were irreversible, the t 1/2 for internalization would be 8.5 hour. Morphometric studies on the uptake of horseradish peroxidase indicate that the t 1/2 for internalization of bulk membrane in granulosa cells is 55 to 77 minutes. Thus, the rate of uptake of surface-bound hCG appears to be seven to nine times slower than the rate of uptake of bulk plasma membrane, which suggests that the LH/hCG receptor may be selectively excluded from the endocytic vesicles of granulosa cells.     

Thyroliberin stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phosphodiesterase in rat mammotropic pituitary cells. Evidence for an early Ca2+-independent action.

Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4'-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4'-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes.     

Clinical strains of Candida albicans express the surface antigen glyceraldehyde 3-phosphate dehydrogenase in vitro and in infected tissues

We have previously described the presence of an enzymatically active form of glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) in the cell surface of Candida albicans ATCC 26555 which is also a fibronectin and laminin binding protein. Immunohistochemical analysis of tissue sections from patients with disseminated candidiasis with a polyclonal antiserum to GAPDH from C. albicans (PAb anti-CA-GAPDH) revealed that the enzyme is expressed at the surface of fungal cells in infected tissues. The same PAb detected the presence of GAPDH species, with a molecular mass of approximately 33 kDa, in cell wall extracts obtained from clinical isolates of the fungus. These cell surface-bound GAPDH moieties exhibited a dose-dependent dehydrogenase activity. These results indicate that this cell surface-bound GAPDH plays a role during infection probably contributing to the attachment of fungal cells to host tissues.     

4,4'-Bis dimethylaminodiphenylcarbinol: a new reagent for selective chemical modification. Interaction with porcine malate dehydrogenase

4,4-bis Dimethylaminodiphenylcarbinol (BDC-OH) has recently been reported to be a highly sensitive reagent for the quantitative determination of sulfhydryl residues in biological materials (1). In this communication the effectiveness of BDC-OH as a reagent for selective chemical modification of “active center” cysteine residues was investigated. The supernatant and mitochondrial forms of malate dehydrogenase were chosen for investigation by this reagent. Supernatant malate dehydrogenase which has never been found to contain an “active center” cysteine is unaffected by this reagent. Mitochondrial malate dehydrogenase (L malate: NAD⁺ oxidoreductase, EC 1.1.1.37) from porcine heart can be irreversibly inactivated by a 20 fold M excess of the reagent. Chemical modification of two essential sulfhydryl residues is prevented by the presence of the coenzyme, NAD⁺, suggesting that the site of interaction is located at or near the coenzyme binding site and hence at or near the enzymatic center of this enzyme.     

Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms

Different chemical methods used to attach oligonucleotides by their 5′-end on a glass surface were tested in the framework of solid phase PCR where surface-bound instead of freely-diffusing primers are used to amplify DNA. Each method was first evaluated for its capacity to provide a high surface coverage of oligonucleotides essentially attached via a 5′-specific linkage that satisfyingly withstands PCR conditions and leaves the 3′-ends available for DNA polymerase activity. The best results were obtained with 5′-thiol-modified oligonucleotides attached to amino-silanised glass slides using a heterobifunctional cross-linker reagent. It was then demonstrated that the primers bound to the glass surface using the optimal chemistry can be involved in attaching and amplifying DNA molecules present in the reaction mix in the absence of freely-diffusing primers. Two distinct amplification processes called interfacial and surface amplification have been observed and characterised. The newly synthesised DNA can be detected and quantified by radioactive and fluorescent hybridisation assays. These new surface amplification processes are seen as an interesting approach for attachment of DNA molecules by their 5′-end on a solid support and can be used as an alternative route for producing DNA chips for genomic studies.     

Reaction between sheep liver mitochondrial aldehyde dehydrogenase and various thiol-modifying reagents.

Sheep liver mitochondrial aldehyde dehydrogenase reacts with 2,2'-dithiodipyridine and 4,4'-dithiodipyridine in a two-step process: an initial rapid labelling reaction is followed by slow displacement of the thiopyridone moiety. With the 4,4'-isomer the first step results in an activated form of the enzyme, which then loses activity simultaneously with loss of the label (as has been shown to occur with the cytoplasmic enzyme). With 2,2'-dithiodipyridine, however, neither of the two steps of the reaction has any effect on the enzymic activity, showing that the mitochondrial enzyme possesses two cysteine residues that must be more accessible or reactive (to this reagent at least) than the postulated catalytically essential residue. The symmetrical reagent 5,5'-dithiobis-(1-methyltetrazole) activates mitochondrial aldehyde dehydrogenase approximately 4-fold, whereas the smaller related compound methyl l-methyltetrazol-5-yl disulphide is a potent inactivator. These results support the involvement of mixed methyl disulphides in causing unpleasant physiological responses to ethanol after the ingestion of certain antibiotics.