Potential of laser immunoassay for detection of HIV in human blood serum and urine.

The potential of Light Scattering Immunoassay (LIA) for detection of HIV in human blood serum has been explored by monitoring the agglutination of antigen coated polystyrene particles by dynamic light scattering. ELISA tested human sera having HIV, TB, Filaria along with normal sera have been analyzed using two specific synthetic peptide antigen (SP1, SP2) and one nonspecific peptide antigen (NSP). Few paired human sera and urine samples and nonspecific (of nonHIV diseases) urine samples have also been tested using the same antigens to check the possibility of replacement of sera by urine.

Design and development of an immunosensor for the detection of malaria in field conditions.

Eradication of malaria in Southeast Asian countries is still a distant goal, due to the absence of a simple, rapid and inexpensive diagnostic technique. Here, an immunosensor for the photometric detection of malaria, the malaria-detecting immunosensor (MDI), is developed to detect Plasmodium falciparum malarial antibodies in human blood. The method uses the principle of laser light-scattering by latex bead agglutinates in media monitored by a light-detecting device. Agglutination is induced by mixing antigen-coated latex beads with serum antibodies. Immunoreactions are measured in terms of the Tyndall effect in the transmitted beam detected by photodiodes. MDI sensitivity and specificity are compared with the results of enzyme-linked immunoabsorbent assay and laser light-scattering immunoassay techniques, which show that it is a good and sensitive monitoring device.

Testing of newly developed glycophospholipid antigen for the detection of P. falciparum malaria by laser light immunoassay in endemic and non-endemic areas.

A glycophospholipid (GPL) antigen isolated from Plasmodium falciparum culture supernatant has been tested for its antigenicity. Detection of malaria positive known blood samples and unknown field samples from endemic and non-endemic areas were compared. In this study laser light scattering immunoassay (LIA) was used for the detection of P. falciparum malaria. Test results of control (malaria negative samples from Surat) were compared with known positive samples and unknown malaria positive field samples. A positive correlation has been observed (97%) in falciparum positive samples from laboratory and unknown samples from endemic area (Haldwani) by LIA method using GPL antigen. From the results of the study it was found that GPL antigen has a better antigenic property and can detect almost all the cases of Pf malaria by LIA method.

Reduced activity of bamhi variants c54i, c64w, and c54d/c64r is consistent with the substrate-assisted catalysis model.

Three specific mutants, C54I, C54W, and a double-mutant C54D:C64R of restriction endonuclease BamHI, were generated and studied to investigate the role, if any, of the 54th and 64th cysteine residues in the catalysis of BamHI. The mutation was achieved using the megaprimer approach for PCR. The mutant genes were cloned and characterized by sequencing. The mutant and the wild-type proteins were expressed and purified and their kinetic parameters were determined using short synthetic oligonucleotides as substrates. All mutants had higher K(m) values than that of the wild-type enzyme suggesting a decrease in the affinity of the enzyme for its substrate. The mutant protein C54W showed significant changes in the CD spectra vis-a-vis wild-type enzyme and had the lowest K(m)/K(cat) value among the mutants indicative of changes in the secondary structure of the protein. The melting curves of the mutant proteins overlapped that of the wild-type enzyme. Analysis of the K(cat) values in the context of cocrystal structure suggests that the effect of Cys54 mutation is probably through the perturbation of the local structure whereas reduced activity of the double mutant is consistent with the substrate-assisted catalysis mechanism.

An alternative approach for screening active bam HI variants: overexpression in T-7 RNA polymerase based system.

The type II restriction endonuclease, Bam HI, has been overexpressed in E. coli by cloning the Bam HI gene in frame with an E. coli Ribosome Binding Site (RBS) under the T7 promoter of an E. coli expression vector pRSET A. The expression level of Bam HI endonuclease using this construct was found to be higher than that reported of the overexpressing clone pAEK14. Our overexpressing clone, pAABRw in BL21 cells in presence of Bam HI methylase in pMAP6 following induction with IPTG yields about 9.2 x 10(6) units per gram wet cell paste. In vivo activity of the recombinant endonuclease could be confirmed by the SOS induction assay in JH139 cells even in the absence of T7 polymerase and cognate Bam HI methylase because of leaky expression in E. coli. This provides an alternate way to screen the active endonuclease and its variants.

Properties and secondary structure analysis of BanI endonuclease: identification of putative active site.

Biochemical properties of Type II restriction enzyme BanI were characterized. Kinetic parameters were evaluated and an enhancement of rate was observed when the recognition site was located in a more central position in the substrate, suggesting that BanI locates its recognition site by a sliding mechanism. As BanI has three cysteine residues in its primary sequence, the effect of thiol inhibitors on BanI activity was also studied. Partial inhibition was observed only at a very high concentration of the inhibitor indicating that cysteine residues are not directly involved in catalysis. The gel electrophoretic mobility shift assay demonstrated specific complex formation between BanI and the DNA substrate in the presence of poly dI-dC and Mg(2+). A secondary structure analysis and comparison with EcoRI and BamHI crystal structure revealed a putative active site similar to that seen in BamHI but different in the order in which the catalytic domain (central beta-sheet) and recognition domain (adjacent alpha-helix) were arranged in the protein.

BanI restriction endonuclease binds in the major groove of DNA: an inhibition kinetic study using substrates with mismatch basepairs.

Structural information on BanI-DNA interaction was obtained from simple inhibition kinetic assays using altered substrates. Self-complementary 13-mer oligodeoxynucleotides with or without mismatch basepairs in the BanI recognition sequence (GGPyPuCC) were synthesized. UV melting curves and CD spectra indicated double-stranded B-DNA structure for all the oligomers. Among the seven oligomers with recognition sequences, GGTACC, GGTGCC, GGTATC, GGCACC, GGAGCC, GGTAAC, and GGATCC, only the first two were cleaved with BanI. Kinetics of BanI cleavage of the native substrate was inhibited competitively by all of the other oligomers except the one with sequence GGCACC. From inhibition kinetic analysis in presence of a fixed concentration of the inhibitor, apparent K(m) and K(I) were determined. The data were analyzed in the context of alterations made in the hydrogen bonding potential in the major and minor groove of DNA within the recognition sequence due to basepair mismatches. Such analyses led to the conclusion that BanI, like BamHI, binds in the major groove and the central thymines make important contact with the protein.

Laser light scattering immunoassay for malaria.

Laser light scattering immunoassay (LIA) was proposed as a prospective diagnostic method for the detection of antibody (or antigen) by monitoring the agglutination of antigen (or antibody) coated carrier particles using dynamic light scattering (DLS) as probe. LIA is a very sensitive assay as it can detect microscopic immune complexes even when antibody (or antigen) level is low. A sizeable number of human sera collected from malaria endemic areas and hospitals have been analysed by ELISA using Pf parasite lysate or a RESA derived synthetic peptide as antigen parallel to LIA using Pf antigen coated polystyrene latex beads. Comparative analysis of data suggests LIA to be as good as ELISA and possibly better in terms of sensitivity and simplicity. LIA can be a simple and inexpensive immunoassay suitable for field use and mass application.

Protein engineering of BamHI restriction endonuclease: replacement of Cys54 by Ala enhances catalytic activity.

Chemical modification studies of BamHI endonuclease indicated the importance of the cysteine residue in catalysis [Nath, K. (1981) Arch. Biochem. Biophys, 212, 611-617]. Of the three cysteine residues at positions 34, 54 and 64 in the BamHI endonuclease Cys54 and Cys64 are at the DNA-protein interface. The co-crystal structure of the BamHI-DNA complex, however, does not indicate any role of cysteines either in binding or catalysis. In the context of strong biochemical evidence, Cys54 in BamHI was changed to Ala54 to investigate its role in catalysis. The mutation was carried out by PCR overlap extension, the mutant gene was cloned and characterized by sequencing. The mutant BamHI was expressed and purified to homogeneity and the kinetic parameters (K(M) and kcat) of the wild type and the C54A mutant were determined. The mutation results in up to approximately 40% enhancement of kcat and some increase in K(M). These in vitro results were also supported by in vivo SOS induction assays: the C54A mutant gene under the T7 promoter caused complete lysis in JH139 in absence of T7 RNA polymerase whereas the wild-type gene gave deep blue colonies under the same conditions. The results suggest no direct role of Cys54 in catalysis, but it can influence the catalytic activity through Val57 backbone contact seen in the co-crystal structure.

Excess nucleoside triphosphates (or zinc) allow recovery of alkaline phosphatase activity following refolding under reducing conditions.

The contribution of ATP and other nucleotides to the stabilization of non-native structures has been described for some proteins. We report here the effect of GTP, ATP, and their nonhydrolyzable analogues on the denaturation and renaturation of the enzyme Escherichia coli alkaline phosphatase. We show that GTP, ATP, and their nonhydrolyzable analogues considerably stimulate renaturation of AP in the presence of 2-mercaptoethanol where spontaneous renaturation is completely arrested due to reduction of S-S bonds. GTP is the most efficient inducer of reconstitution of the active site and appears to play a specific role besides being a substrate. The reconstituted protein was found to be in the reduced form despite having near-normal activity. The self-refolded oxidized form and the GTP-refolded reduced form had the same KM/kcat values and showed similar structural properties. We conclude that GTP can not only induce reconstitution of dimerization-competent monomers because of its substrate nature but also act as a modulator of the activity of AP. We also report here on the Zn2+-assisted reconstitution of E. coli AP under reducing condition. The prior formation of a disulfide bond for positioning the active site residues in the proper geometry is unnecessary under this condition.

Laser light scattering immunoassay: an improved data analysis by CONTIN method.

Laser light scattering immunoassay (LIA) is a diagnostic method for the detection of antibody by monitoring the agglutination of antigen carrier particles mediated by antibody, using dynamic light scattering (DLS) as probe. We have used this method for the detection of antibody to P. falciparum that cause malaria. The data were analysed using CONTIN method and the superiority of the distribution analysis over the conventional interpretation of the data in terms of mean diffusion coefficient or hydrodynamic radius is discussed in detail.

DNA recognition and structural specificities.

How a short DNA sequence interacts in a sequence specific manner with appropriate protein is understood only in certain systems for which high resolution crystal structures of the protein-DNA complexes are available. The base sequence of DNA is sensed directly (read-out) by the protein through the major or minor groove, while DNA shape also is sensed through multiple interactions with the sugar phosphate backbone. Several repressors, activators and restriction endonucleases complexed with their cognate DNA oligomers are now known and reviewed here. If the binding site on DNA has two fold symmetry, the protein interacts as dimer and uses a variety of structural motifs for specific interaction. The level of specificity of interaction is enhanced by flexibility and/or distortion in either the DNA or protein tertiary structure.

Bam HI cleaves the self complementary dodecamer d-CGCGGAGCCGCG, before the two G's and possibly binds in the DNA major groove.

Oligodeoxynucleotides with GT or GA mispairs within the Bam HI recognition sequence (GGATCC), have been prepared. Binding and cleavage of the native vis a vis the mismatch substrates by Bam HI are analysed. UV melting curves and CD spectra of the oligomers suggest a double stranded B-DNA conformation. The enzyme Bam HI binds with varying affinities to the oligomers except the one with the GT wobble base pair. Bam HI cleaves the cognate sequence, GGATCC, between the two Guanines but cleaves GGAGCC before the guanines. The unusual cleavage is due to a local distortion in the DNA structure. Kinetic analysis of the cleavage reactions using the 35S labeled hexadecamers, d-ATGGCGGATCCGCCAT and d-ATGGCGGAGCCGCCAT, as substrates gives Km values 11.08 nM and 1.16 nM with corresponding Kcat of 11.04 and 0.62 min-1 respectively. The results are consistent with the binding of Bam HI in the major groove.

Use of isotope-dilution phenomenon to advantage in the determination of kinetic constants Km and Kcat for BamHI restriction endonuclease: an empirical and iterative approach.

An assay using a very small amount of 35S-labeled deoxyoligonucleotide as a substrate for the determination of Km and Kcat for the restriction enzyme BamHI is described. Two synthetic deoxyoligonucleotides, ATGGCGGATCCGC and ATGGCGGAGCCGC, containing the cognate and a mismatch BamHI sequence, respectively, were labeled by an end-filling reaction using the Klenow fragment of DNA polymerase and [35S]dATP to generate the labeled self-complementary substrates. The dependence of BamHI hydrolysis on substrate concentration was investigated using mixtures of a fixed amount of radiolabeled substrate and varying amounts of cold-labeled substrate over a wide range. The apparent competitive inhibition observed due to the phenomenon of carrier dilution was analytically corrected by an empirical as well as an iterative approach to give Km values comparable to those reported in the literature. We have found that the values obtained using the empirical formula are very close to the precise values obtained through iteration. Our procedure has used isotopic dilution to advantage to make the assay less expensive and can be applied effectively to any enzyme-substrate reaction in which the substrate and the product have radioactive labels. The method would be especially useful for a rapid analysis and comparison of kinetic constants of various mutant enzymes or substrates.

Hairpin formation in d-AAGCTTAAGCTT under high salt conditions shows unusual properties.

The hairpin-duplex equilibria of the dodecamer d-AAGCTTAAGCTT and interaction of the duplex form with a pentapeptide, KGWGK, has been studied. UV thermal transitions are monophasic at low salt but biphasic at higher salt concentrations. At 10(-5) M or less oligomer concentration biphasic melting curves persist till 900 mM NaCl. The d(Tm)/d log(Na+) for the duplex form is 12 degrees C and for the hairpin is 18 degrees C. The delta H and delta S values for duplex formation are low (-25 K cal/mole and -59 Cal/mole respectively). KGWGK binds to the duplex form with a binding constant K = 3.4 x 10(5)M-1 measured from fluorescence quenching of tryptophan. These unusual results are markedly different from that reported for d-AGATCTAGATCT (Biochemistry 31, 6241-6245) and are discussed in terms of sequence dependence of loop folding and cruciform extrusion pathway of hairpin formation.

Binding of oligopeptides to d-AGATCTAGATCT and d-AAGCTTAAGCTT: can tryptophan intercalate in DNA hairpins?

The interactions of three tryptophan-containing peptides, KWK, KGWK tert-butyl ester, and KGWGK, with two self-complementary dodecamers of the same base composition but different sequence were studied by UV, CD, and fluorescence spectroscopy. The oligonucleotides, d-AGATCTAGATCT and d-AAGCTTAAGCTT, contain tandem repeats of the recognition site for the restriction enzyme BglII in the former and HindIII in the latter. Thermal transition data in dilute solutions and in 0.01 M NaCl indicate these dodecamers to be present in hairpin forms. Binding of peptides to these hairpins was followed by tryptophan fluorescence quenching titrations at 10 mM Na+; the data suggest intercalation of the indole ring. The association constants for the peptide-oligonucleotide (PN) complexes are an order of magnitude higher (10(5) M) than those reported with polynucleotides [10(4) M; Rajeswari et al. (1987) Biochemistry 26, 6825]. The pentapeptide, KGWGK, discriminates between BglII and HindIII sequences with higher affinity for the HindIII dodecamer. The CD maximum of KGWGK, at 220 nm, is drastically diminished upon interaction with oligonucleotides. The ellipticity at 220 nm is halved at 10 times less P/N ratio with the HindIII dodecamer than the BglII dodecamer, suggesting stronger binding to the HindIII dodecamer. The results are discussed in terms of two different modes of binding of oligopeptides to the DNA hairpins.

Hairpin and duplex forms of a self-complementary dodecamer, d-AGATCTAGATCT, and interaction of the duplex form with the peptide KGWGK: can a pentapeptide destabilize DNA?

Ordered forms of a synthetic dodecamer, d-AGATCTAGATCT, a direct repeat of the BglII recognition sequence, have been investigated using UV, CD, and fluorescence spectroscopy. Complex hairpin-duplex equilibria are manifest in UV thermal transitions, which are monophasic in the presence of very low or high NaCl concentrations but distinctly biphasic at intermediate ionic strengths. In 100 mM NaCl, the 1/Tm vs 1n C curve has a reasonable positive slope, which yields delta H and delta S for duplex formation as -66.2 kcal/mol and -190 cal/mol, respectively. Interaction of the dodecamer in duplex form with a tryptophan-containing peptide, KGWGK, has also been investigated to test the "bookmark" hypothesis (Gabbay et al., 1976) under the uniform structural constraint of the oligonucleotide of defined sequence. CD spectra of the peptide (P), the oligonucleotide (N), and their mixtures at different P/N ratios show a dramatic change in peptide spectrum but little change in nucleic acid dichroism with peptide binding. The Tm of P-N complexes decreases with an increase in peptide binding and levels off at saturation binding of P/N = 2.0. The data are interpreted in terms of a groove-cum-intercalation mode of binding, where intercalation to the tryptophan side chain destabilizes the double helix. A Scatchard plot of the binding data is nonlinear, with best-fit values for an overall association constant K = 4.33 x 10(5) M-1, and the number of binding sites n = 3.23 when fitted to the site-exclusion model of binding.

Fast and simple anion-exchange chromatography for large-scale purification of self-complementary oligonucleotides.

A fast and simple anion-exchange chromatography method is described for large-scale purification of synthetic oligonucleotides. Using a single matrix and aqueous solvent system, the two-step chromatographic procedure can handle complex separation problems of self-complementary or G-rich sequences without the use of urea or formaldehyde. The work also demonstrates the complication encountered, possibly due to hairpin formation, in one of the oligomers.