Direct measurement of diffusion rates in enzyme crystals by video absorbance spectroscopy

Abstract

In order to directly observe the structure of enzyme intermediate complexes that form during turnover, one must ensure the homogeneous and complete accumulation of the intermediate during the collection of diffraction data. In the case of * Author to whom all correspondence should be addressed.:~:~ 1995 International Union of Crystallography Printed in Great Britain -all rights reserved steady-state Laue experiments, the accumulation of the intermediate is dictated by the rate of substrate diffusion throughout the crystal, compared with the rate of turnover and disappearance of a rate-limited catalytic intermediate. This paper describes a simple quantitative method for measuring substrate diffusion and binding within an enzyme crystal. The set-up consists of a light source, specific bandpass filters, a crystallographic flow cell, a syringe pump, a charge-coupleddevice color video camera and a workstation with a frame- Journal of Applied Crystallography

Quantifying anisotropic solute transport in protein crystals using 3‐D laser scanning confocal microscopy visualization

“…Granick (1942) determined transport parameters of solutes in protein crystals qualitatively by monitoring the overall color variations in a guinea pig oxyhemoglobin crystal due to the penetration of K-ferricyanide. The quantitative determination of transport parameters commenced with the introduction of X-ray fluorescence measurements (Bishop and Richards, 1968), video absorbance spectroscopy (O'Hara et al, 1995), and quantitative fluorescence microscopy (Velev et al, 2000). These analytical methods allow the determination of accumulated concentration profiles through the protein crystal.…”

A microspectrophotometer for UV–visible absorption and fluorescence studies of protein crystals

“…For such studies, it is an advantage to be able to follow substrate binding, product release, and accumulation of intermediates within the crystal prior to exposure to X-rays. Analytical methods that have been used include high-pressure liquid chromatography (Schlichting et al, 1989), mass spectroscopy (Cohen & Chait, 2001), video absorption spectroscopy (O'Hara et al, 1995) or absorption microspectrophotometry (Mozzarelli & Rossi, 1996, and references therein).…”

Advances in spectroscopic methods for biological crystals. 1. Fluorescence lifetime measurements

“…For such studies, it is an advantage to be able to follow substrate binding, product release, and accumulation of intermediates within the crystal prior to exposure to X-rays. Analytical methods that have been used include high-pressure liquid chromatography (Schlichting et al, 1989), mass spectroscopy (Cohen & Chait, 2001), video absorption spectroscopy (O'Hara et al, 1995) or absorption microspectrophotometry (Mozzarelli & Rossi, 1996, and references therein).…”