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

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 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‐coupled‐device color video camera and a workstation with a frame‐grabber card for collection and digitization of images and subsequent processing of data. By this technique, any diffusion and binding process in a protein crystal leading to a visible absorbance shift may be accurately monitored and quantified during data collection. This method offers the crystallographer an inexpensive and simple alternative to the use of a single‐crystal microspectrophotometer to measure the relatively slow process of diffusion.