Micro-volume couette flow sample orientation for absorbance and fluorescence linear dichroism

Abstract

Linear dichroism (LD) can be used to study the alignment of absorbing chromophores within long molecules. In particular, Couette flow LD has been used to good effect in probing ligand binding to DNA and to fibrous proteins. This technique has been previously limited by large sample requirements. Here we report the design and application of a new micro-volume Couette flow cell that significantly enhances the potential applications of flow LD spectroscopy by reducing the sample requirements for flow linear dichroism to 25 microL (with concentrations such that the absorbance maximum of the sample in a 1-cm pathlength cuvette is approximately 1). The micro-volume Couette cell has also enabled the measurement of fluorescence-detected Couette flow linear dichroism. This new technique enables the orientation of fluorescent ligands to be probed even when their electronic transitions overlap with those of the macromolecule and conversely. The potential of flow-oriented fluorescence dichroism and application of the micro-volume Couette LD cell are illustrated by the collection of data for DNA with minor groove and intercalating ligands: DAPI, Hoechst, and ethidium bromide. As with conventional fluorescence, improved sensitivity compared with absorbance LD is to be expected after instrumentation optimization.

FIGURE 1

Schematic diagram of LD flow Couette cell.

FIGURE 2

Schematic diagram showing the capillary and rod assembly in the micro-volume Couette cell.

FIGURE 3

(a) Photograph of micro-volume Couette flow cell and (b) schematic diagram of micro-volume Couette flow cell.

FIGURE 4

General arrangement drawing showing detail of quartz micro-volume Couette flow LD cell.

FIGURE 5

A chart to show the relationship between voltage and rpm of the capillary.

FIGURE 6

(a) LD spectrum of ct-DNA (550 μM) in sodium cacodylate buffer (10 mM, pH 7) and NaCl (10 mM); (b) LD₂₅₉ versus DNA concentration in a capillary cell at voltage 4 V.

FIGURE 7

LD spectra of DNA (200 μM) and different concentrations of ethidium bromide (0–50 μM) using a sodium cacodylate buffer (10 mM, pH 7) and NaCl (10 mM).

FIGURE 8

Spectra of DNA (1000 μM) and DAPI (50 μM). (a) Absorption spectra of DAPI (dashed line) and DAPI-ct-DNA (solid line), (b) fluorescence excitation spectrum of DAPI-ct-DNA with all emitted photons detected, (c) LD spectra of ct-DNA (dashed line) and DAPI-ct-DNA (solid line), and (d) FDFLD spectrum of DAPI-ct-DNA. All solutions prepared using a sodium cacodylate buffer (10 mM, pH 7).

FIGURE 9

Spectra of DNA (1000 μM) and Hoechst (50 μM). (a) Absorption spectra of Hoechst (dashed line) and Hoechst-ct-DNA (solid line), (b) fluorescence excitation spectrum of Hoechst-ct-DNA with all emitted photons detected, (c) LD spectra of ct-DNA (dashed line) and Hoechst-ct-DNA, and (d) FDFLD spectrum of Hoechst-ct-DNA. All solutions prepared using a sodium cacodylate buffer (10 mM, pH 7).

FIGURE 10

DNA (1000 μM) and ethidium bromide (50 μM). (a) Absorption spectra of ethidium bromide (dashed line) and ethidium bromide-ct-DNA (solid line). (Inset) Enlarged region of ethidium bromide band. (b) Fluorescence excitation spectra with all emitted photons detected, (c) LD spectra of ct-DNA (dashed line) and ethidium bromide-ct-DNA (solid line), and (d) FDFLD spectra. All solutions prepared using a sodium cacodylate buffer (10 mM, pH 7).

FIGURE 11

Plot of from 0° to 180°, although the plot has symmetry ∼90°.