Challa V. Kumar

Groove binding of a styrylcyanine dye to the DNA double helix : the salt effect

Abstract The cationic styryl dye DSMI binds to the double helical DNA with a high affinity. A red shift of the absorption spectrum and hypochromism accompany the binding of DSMI to calf thymus DNA. The fluorescence yields increase dramatically when DSMI binds to DNA, with no shifts in the emission maximum. These spectral changes are in contrast to the behavior observed with many fluorescent intercalators. Groove binding rather than intercalation of the dye was suggested to be the cause of these spectral changes, based on a variety of experimental results. Consistent with groove binding, the fluorescence enhancements were greater with poly(dA—dT) when compared with poly(dG—dC). The addition of salt or a strong electrolyte to the solution releases the DNA-bound dye cations from the groove and causes a decrease in the fluorescence yield. Therefore, when the dye binds to DNA, the fluorescence quenching with anionic quenchers was enhanced dramatically, in contrast to the expected protection of the probe fluorescence by the DNA helix. Binding of DSMI to DNA inhibits the non-radiative deactivation of the excited state, perhaps owing to the restriction imposed by the narrow minor groove of the DNA. The steric constraints imposed on the dye owing to the binding in the groove result in enhanced fluorescence yields and longer fluorescence lifetimes. Since the lifetimes of the DNA-bound dye were still in the nanosecond range, it is very likely that groove binding of the dye permits twisting in the excited state, which may be important in the excited state dynamics of this class of dyes. The large fluorescence enhancements observed when DSMI binds to the helix and the poor fluorescence yield of the dye in the absence of DNA suggests that DSMI should be quite useful in the staining of DNA gels and for the detection of very low concentrations of DNA.

Luminescence of ruthenium(II) polypyridyls: evidence for intercalative binding to Z-DNA

Photophysical studies have been undertaken to characterize the binding interactions of enantiomers of Ru(phen)3(2+), Ru(DIP)3(2+), and racemic Ru(bpy)2dppz2+ (where phen = 1,10-phenanthroline, DIP = 4,7-diphenylphenanthroline, and dppz = dipyridophenazine) with Z-form poly d(GC). Parallel enhancements in steady state luminescent intensity and a lengthening of luminescent lifetimes are seen for ruthenium enantiomers with Z-DNA as for B-DNA but with enantioselectivities reversed. Greater enhancements are seen for delta-isomers with the right-handed helix but for lambda-isomers with the left-handed helix. Ru(bpy)2dppz2+, an avid intercalator in B-DNA, displays no luminescence free in aqueous solution, but luminesces brightly bound to either B- or Z-poly d(GC). Stern-Volmer quenching studies also support the enantioselective preference in binding to B-DNA by delta-isomers and a reversal with binding to Z-DNA preferentially by the lambda-isomers. Steady state polarization studies indicate a rigid association of the complexes with both B- and Z-DNA on the time-scale of their emission and again with symmetrical enantioselectivities for the left and right-handed helices. Given the well characterized intercalative association of the complexes with B-DNA, the parallel results seen here with Z-DNA point strongly to a comparable intercalative association with the Z-form helix. That molecules may interact with Z-DNA through intercalation has not been demonstrated previously and now requires consideration in describing the range of interactions of small molecules and proteins with Z-DNA.

Attomolar Detection of a Cancer Biomarker Protein in Serum by Surface Plasmon Resonance Using Superparamagnetic Particle Labels

Methods to measure protein biomarkers with ultralow detection limit (DL) and high sensitivity promise to provide valuable tools for early diagnosis of diseases such as cancer, and for monitoring therapy and post-surgical recurrence.[i, ii] Surface plasmon resonance (SPR) utilizing nanoparticle-antibody labels for signal amplification in immunoassays is an emerging approach for detecting proteins in biomedical samples.[iii–x] Herein, we show for the first time that clustering of superparamagnetic labels on SPR surfaces leads to unprecedented sensitivity and ultralow DL for protein biomarkers in serum. Specifically, antibody bioconjugates on 1 µm diameter superparamagnetic particles (MP) used for off-line antigen capture enabled SPR detection of cancer biomarker prostate specific antigen (PSA) in serum at an ultralow DL of 10 fg mL−1 (~300 aM). This approach opens doors for accurate diagnostics based on new protein biomarkers with inherently low concentrations.

Aromatic thioketone triplets and their quenching behaviour towards oxygen and di-t-butylnitroxy radical. A laser-flash-photolysis study

Results obtained using nanosecond laser flash photolysis with various exitation wavelengths (337.1–600 nm) are reported for triplet-related properties of five aromatic thioketones, namely xanthione, thioxanthione, thiobenzophenone and p,p′-dimethoxy and p,p′-bis(N,N-dimethylamino) derivatives of thiobenzophenone. The triplet yields (ϕT) of these thioketones in benzene are high (0.5–1.0) and show a dependence on excitation wavelengths. The intersystem-crossing efficiency is less than unity (0.5–0.6) when laser excitation is carried out into the second excited singlet state, S2(λex= 355 and 425 nm), but approaches unity when the excitation leads to absorption into the lowest excited singlet, S1(532 and 600 nm). The intrinsic triplet lifetimes are short (0.8–1.8 µs) and the self-quenching rate constants are in the range (2.6–7.1) 109 dm3 mol–1 s–1. Quantitative data concerning triplet–triplet absorption spectra and triplet quenching by azulene, ferrocene, β-carotene and 2,5-dimethyl-2,4-hexadiene are presented. The oxygen quenching rate constants [(2.8–9.7)× 109 dm3 mol–1 s–1] increase when electron-donating groups (methoxy and N,N-dimethyl-amino) are present in the thiobenzophenones, suggesting that charge-transfer interaction is important. The efficiency of singlet-oxygen generation in the course of the oxygen quenching of the p,p′-dimethoxythiobenzophenone triplet is unity. The stable free radical, di-t-butyl-nitroxide, quenches the thioketone triplets with unusually high rate constants [(1.4–3.3)× 109 dm3 mol–1 s–1]; this behaviour appears to be a manifestation of electron-exchange-mediated intersystem crossing (S0 [graphic omitted] T1), enhanced by strong spin–orbit coupling or spin–spin interaction associated with the thiocarbonyl sulphur atom.

Substituent effects in the quenching of acetophenone and benzophenone triplets by oxygen and the di-tert-butylnitroxy radical, and the efficiency of singlet oxygen photogeneration

Abstract The behavior of the triplets of a number of p-substituted acetophenones and benzophenones with respect to quenching by oxygen and the di-tert-butylnitroxy radical (DTBN) has been examined in benzene and acetonitrile using 337.1 nm laser flash photolysis. In several cases the efficiency φΔ of singlet oxygen (1O2*) generation in the course of triplet quenching by oxygen has been determined using 1,3-diphenylisobenzofuran as the trapping agent for 1O2*. The variation of the rate constants for triplet quenching with respect to the substituent nature suggests that the aromatic carbonyl triplets act as donors and acceptors in the quenching interaction with oxygen and DTBN respectively. Relative to the rate constant for quenching by oxygen, the substituent effect on φΔ is less pronounced, particularly in the case of benzophenones; the φΔ are limited to the range 0.3 − 0.7 in the two solvents. Fast equilibration among the various spin configurations of the encounter complex responsible for the nearly substituent-independent φΔ for benzophenones and back electron transfer in charge-transfer-derived ion pairs leading to 1O2* formation are suggested.

DNA-based supramolecular artificial light harvesting complexes.

Solar radiation reaching this planet is distributed over a wide range of wavelengths, and efficient collection and conversion of solar energy requires light harvesting over multiple wavelengths. Yet, the design, synthesis, and testing of novel, efficient, inexpensive light harvesting complexes are lacking. Engineered protein-DNA complexes are used here to self-assemble donor and acceptor molecules into artificial light harvesting units with an association constant of 3.3 +/- 1.2 muM(-1). Excitation of the DNA-bound donors resulted in a 540% increase in emission from the protein-bound acceptors, and the presence of one acceptor for each pair of donors was sufficient to quench approximately 50% of donor emission. Successful self-assembly of DNA-based light harvesting units is expected to facilitate economic/efficient conversion of solar energy, and model systems to achieve this goal are demonstrated here. We anticipate that success along these lines would facilitate more efficient approaches for solar energy capture.

Chiral protein scissors: High enantiomeric selectivity for binding and its effect on protein photocleavage efficiency and specificity

Chiral recognition of protein-binding sites by a simple organic molecule with selectivities >100 is reported here. The l-isomer of 4(1-pyrene)-3-butyroyl-phenylalanine amide (Py-l-Phe) binds to BSA with an affinity constant (Kb) of 3 × 107 M−1, whereas the corresponding d-isomer (Py-d-Phe) binds 100 times weaker. The enantiomers showed contrasting spectral changes when bound to BSA. Whereas hypochromism was observed with the l-isomer, hyperchromism was observed for the d-isomer, and, whereas the fluorescence of the l-isomer was quenched, the fluorescence of the d-isomer was enhanced. The induced CD spectra of the enantiomers bound to BSA bear a near mirror-image relationship. In contrast, the enantiomers show only moderate binding selectivity with lysozyme. The differences in the enantioselectivities with the two proteins indicate that the binding site of 4(1-pyrene)-3-butyroyl-phenylalanine amide (Py-Phe) in BSA is crowded, whereas that of lysozyme is more accommodative of either isomer. The enantioselective binding of Py-Phe isomers is further examined in protein photocleavage studies. Py-d-Phe cleaves BSA and lysozyme at a single site in a manner similar to Py-l-Phe, but the cleavage yields are lower for the d-isomer. Sequencing of the resulting fragments indicated that the photocleavage sites of Py-d-Phe on BSA and lysozyme are identical to those of Py-l-Phe. Flash photolysis studies indicated only minor differences between the two enantiomers. The large binding selectivities, therefore, do not influence cleavage specificity or cleavage site location. The strong role of the single asymmetric center of Py-Phe in recognition and its minor role in photocleavage chemistry are demonstrated.

The role of cinchona alkaloids in enantioselective hydrogenation reactions: Are they modifiers or hosts involved in supramolecular heterogeneous catalysis?

Abstract In this study experimental evidences are summarized supporting the modifier–substrate interaction taking place in the liquid phase in the enantioselective hydrogenation of α-keto esters and related compounds. The results indicate that the catalytic system cinchona alkaloids-supported platinum (or palladium) can effectively be used in enantioselective hydrogenation for prochiral substrates, in which the prochiral group is part of a conjugated double bond system. It is considered that the above catalytic system is the first example of a new class of heterogeneous catalytic reactions with the involvement of supramolecular catalysis.

Protein polymer conjugates: improving the stability of hemoglobin with poly(acrylic acid).

The synthesis, characterization, and evaluation of a novel polymer-protein conjugate are reported here. The covalent conjugation of high-molecular weight poly(acrylic acid) (PAA) to the lysine amino groups of met-hemoglobin (Hb) resulted in the covalent conjugation of Hb to PAA (Hb-PAA conjugate), as confirmed by dialysis and electrophoresis studies. The retention of native-like structure of Hb in Hb-PAA was established from Soret absorption, circular dichroism studies, and the redox activity of the iron center in Hb-PAA. The peroxidase-like activities of the Hb-PAA conjugate further confirmed the retention of Hb structure and biological activity. Thermal denaturation of the conjugate was investigated by differential scanning calorimetry and steam sterilization studies. The Hb-PAA conjugate indicated an improved denaturation temperature (T(d)) when compared to that of the unmodified Hb. One astonishing observation was that polymer conjugation significantly enhanced the Hb-PAA storage stability at room temperature. After 120 h of storage at room temperature in phosphate-buffered saline (PBS) at pH 7.4, for example, Hb-PAA retained 90% of its initial activity and unmodified Hb retained <60% of its original activity under identical conditions of buffer, pH, and temperature. Our conjugate demonstrates the key role of polymers in enhancing Hb stability via a very simple, efficient, general route. Water-swollen, lightly cross-linked, stable Hb-polymer nanogels of 100-200 nm were produced quickly and economically by this approach for a wide variety of applications.

High temperature peroxidase activities of HRP and hemoglobin in the galleries of layered Zr(IV)phosphate.

Horseradish peroxidase, and met hemoglobin, when intercalated in the galleries of alpha-Zr(IV) phosphate, show peroxidase activities at elevated temperatures (86-90 degrees C) and the rates increased to 2-3.6 times the rates observed at room temperature.