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Preparation and Preliminary Characterization of Crystallizing Fluorescent Derivatives of Chicken Egg White LysozymeFluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins are intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein molecule. Lucifer yellow and 5-(2-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS) have been attached to the side chain carboxyl of Asp(sup 101) using a carbodiimide coupling procedure. Asp(sup 101) lies within the active site cleft, and it is believed that the probes are "buried" within that cleft. Lucifer yellow and MANS probes with iodoacetamide reactive groups have been bound to His(sup 15), located on the "back side" of the molecule relative to the active site. All the derivatives fluoresce in the solution and the crystalline states. Fluorescence characterization has focused on determination of binding effects on the probe quantum yield, lifetime, absorption and emission spectra, and quenching by added solutes. Quenching studies show that, as postulated, the Asp(sup 101)-bound probes are partially sheltered from the bulk solution by their location within the active site cleft. Probes bound to His(sup 15) have quenching constants about equal to those for the free probes, indicating that this site is highly exposed to the bulk solution.
Document ID
20010086592
Acquisition Source
Marshall Space Flight Center
Document Type
Preprint (Draft being sent to journal)
Authors
Sumida, John
(NASA Marshall Space Flight Center Huntsville, AL United States)
Forsythe, Elizabeth L.
(NASA Marshall Space Flight Center Huntsville, AL United States)
Pusey, Marc L.
(NASA Marshall Space Flight Center Huntsville, AL United States)
Whitaker, Ann F.
Date Acquired
August 20, 2013
Publication Date
January 18, 2001
Subject Category
Atomic And Molecular Physics
Distribution Limits
Public
Copyright
Work of the US Gov. Public Use Permitted.

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