12 research outputs found
Spectrophotometry of crotamine-DNA mixtures in 0.02 M Hepes, pH 7.5, 0.01 M NaCl, 0.0001 M EDTA (standard buffer with indicated [NaCl]).
<p>A. Spectra of 4.1Γ10<sup>β5</sup> M ds calf thymus DNA +4.3Γ10<sup>β6</sup> M crotamine. ββββββ, DNA alone; Β· Β· Β· Β· Β·, crotamine alone; β β β β, DNA + crotamine prior to centrifuging; β Β· β Β· β Β· β, DNA + crotamine supernatant after centrifuging; β Β· Β· β Β· Β· β Β· Β· β, sum of individual DNA and crotamine spectra. B. Spectra of 2.5Γ10<sup>β5</sup> M(p) 25-mer (CCG)<sub>8</sub>C and 2.5Γ10<sup>β6</sup> M crotamine. ββββββ, DNA alone; Β· Β· Β· Β· Β·, crotamine alone; β β β β, DNA + crotamine. C. Titration of ds calf thymus DNA with crotamine; starting [DNA]β=β2.22Γ10<sup>β5</sup> M(p). D. Titration of d(CCG)<sub>8</sub>C with crotamine; starting [DNA]β=β1.21Γ10<sup>β4</sup> M(p). E. Titration of d(CCG)<sub>8</sub>Cβ’G(CGG)<sub>8</sub> with crotamine; starting [DNA]β=β6.2Γ10<sup>β5</sup> M(p).</p
Dependence of Crotamine - d(CCG)<sub>8</sub>C Association Constants on [NaCl]<sup>*</sup>.
*<p>Titrations were performed in 0.02 M Hepes, pH. 7.7, 0.0001 M Na<sub>2</sub>EDTA, and the indicated concentration of NaCl. The [Na<sup>+</sup>] includes the contribution from the buffer (0.01 M).</p
Possible model for crotamine β double stranded DNA major groove interaction.
<p>Crotamine (1h50.pdb) and DNA (1bna.pdb) were generated using Rasmol. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048913#pone.0048913-Sayle1" target="_blank">[46]</a> Sidechains of potentially interacting arginine and tryptophan residues are indicated. The locations of the six lysine Ξ΅-NH<sub>2</sub> groups visible in the pictured orientation are indicated by circles.</p
Binding of d(CCG)<sub>8</sub>C to crotamine as a function of [NaCl].
<p>A. Fluorescence titrations were performed in the standard buffer (0.02 M Hepes, pH 7.5, 0.0001 M EDTA) with the following [NaCl]: βͺ, 0.01 M; β¦, 0.05 M; β‘, 0.075 M; β΄, 0.1 M. B. Reversal of 0.01 M titration by addition of aliquots of a concentrated solution of NaCl. [Na+] was calculated from the [NaCl] and the contribution of the other components of the buffer. The lines connect the points and are shown for clarity.</p
Dependence of binding affinity on oligonucleotide sequence.
<p>A. Fluorescence titration in the standard buffer with 0.01 M NaCl: β―, d(ATGTGGAAAATCTCTAGCAGT) (21+); β΄, d(ACTGCTAGAGATTTTCCACAT) (21-); βͺ, duplex (21+/β). B. NaCl-induced reversal.</p
Binding of d(CCG)<sub>8</sub>C to Arg-Trp-Arg-Trp-Lys-Leu-NH<sub>2</sub> as a function of [NaCl].
<p>A. Fluorescence titrations were performed as in Fig. 3, with the following [NaCl]: βͺ, 0.01 M; β¦, 0.05 M; β‘, 0.075 M; β΄, 0.1 M. B. NaCl reversal: βͺ, reversal of 0.01 M titration of Arg-Trp-Arg-Trp-Lys-Leu-NH<sub>2</sub> with d(CCG)<sub>8</sub>C<sub>;</sub> β―, reversal of 0.01 titration of crotamine with d(CCG)<sub>8</sub>C (data from Fig. 3B).</p
DNA length, concentration, and salt dependencies of crotamine-DNA aggregations, in the standard buffer, unless otherwise indicated.
<p>A. Effect of DNA length. Lane 1: DNA ladder; lane 2βΆ1 Β΅g (3 nmol in residue) DNA ladder +0.3 nmol crotamine, [crotamine]:[DNA]<sub>p</sub>β=β1βΆ10; lane 3: same as lane 2, but treated with SDS (0.1% net) prior to electrophoresis; lane 4: supernatant of mixture as in lane 2 after centrifugation at 13,000 rpm for 5 min; lane 5: precipitate of mixture in lane 4 after treatment with 0.1% SDS. B. Effect of relative DNA concentration. Each lane contained 0.5 Β΅g DNA ladder. Samples were centrifuged at 13,000 rpm for 5 min, and supernatants applied to the gel. Lane 1: DNA alone. [crotamine]:[DNA]<sub>p</sub>β=β1βΆ30 (lane 2),β=β1βΆ20 (lane 3),β=β1βΆ15 (lane 4),β=β1βΆ12 (lane 5),β=β1βΆ10 (lane 6),β=β1βΆ7.5 (lane 7). C. Effect of salt. Each lane contained 1 Β΅g DNA ladder. Lane 1: DNA ladder alone. Lanes 2β7βΆ0.5 Β΅g DNA ladder +0.3 nmol crotamine in 0.01 M NaCl (lane 2: supernatant, lane 3: SDS-treated precipitate), in 0.05 M NaCl (lane 4: supernatant, lane 5: SDS-treated precipitate), in 0.1 M NaCl (lane 6: supernatant, lane 7: SDS-treated precipitate).</p
Scatchard plots and salt dependence of the data in Fig. 3.
<p>A. Scatchard plot: Ξ½ β‘ [crotamine]<sub>bound</sub>/[total d(CCG)<sub>8</sub>C]<sub>p</sub>, L β‘ [crotamine]<sub>free</sub>. The solid lines represent curves calculated from the best fit of the Tsodikov et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048913#pone.0048913-Tsodikov1" target="_blank">[35]</a> modification of the McGhee-von Hippel model for non-cooperative binding ligands <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048913#pone.0048913-McGhee1" target="_blank">[34]</a> (see Materials and Methods). B. Log-log dependence of the association constants calculated in panel A on [Na<sup>+</sup>].</p
Comparison of binding of a single-stranded oligonucleotide (d(CCG)<sub>8</sub>C) to crotamine with the binding of a double-stranded oligonucleotide (d(CCG)<sub>8</sub>Cβ’G(CGG)<sub>8</sub>).
<p>A. Fluorescence titration in the standard buffer with 0.01 M NaCl: β‘, d(CCG)<sub>8</sub>C); β΄, d(CCG)<sub>8</sub>Cβ’G(CGG)<sub>8</sub>. B. NaCl-induced reversal.</p
Effect of oligonucleotide length on binding affinity.
<p>A. Fluorescence titration in the standard buffer with 0.01 M NaCl: βͺ, dT<sub>7</sub>; β΄, dT<sub>14</sub>, β―, dT<sub>21.</sub> B. NaCl-induced reversal.</p