Plurality of pressure-denatured forms in chymotrypsinogen and lysozyme

Journal Article · Tue Dec 14 04:00:00 EST 1976 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00670a023· OSTI ID:7231996

Li, T M; Hook, III, J W; Drickamer, H G; Weber, G

The ultraviolet fluorescence of lysozyme shows a two-step decrease in efficiency when the pressure of the solution is raised from 10/sup -3/ to 11 kbars. The fluorescence of chymotrypsinogen decreases smoothly in a single step confined to the region below 8 kbars, with a midpoint at 5.8 kbars. At 11 kbars, lysozyme binds 1 mol of 8-anilino-1-naphthalenesulfonate (ANS) with a dissociation constant = 4.8 ..mu..M. fluorescencehymotrypsinogen binds 2 mol with a dissociation constant = 14 ..mu..M and Hill coefficient close to unity. In both proteins, binding of ANS is confined to the region above 6 kbars. In lysozyme it roughly corresponds to the second step of ultraviolet fluorescence changes. In chymotrypsinogen it has only minimal overlap with the region of protein-fluorscence change. The existence of two distinct regions of change of ultraviolet fluorescence in lysozyme and the disparity of the regions of change of protein fluorescence and of ANS binding in the two cases demonstrate the existence of a plurality of pressure-denatured forms in both proteins. On addition of tri-N-acetyl-D-glucosamine ((N--Ac--GlcN)/sub 3/) to lysozyme, the two-step change of fluorescence with pressure is replaced by a smooth change in a single step with a midpoint at 5.3 kbars. Analysis of the reciprocal effects expected between ligand binding and denaturation indicates that (N--Ac--GlcN)/sub 3/ binds preferentially to the denatured form of the protein. The standard free energy of binding to this form is estimated to be some 3 kcal/mol higher than the free energy of binding to the native enzyme. Computation of the spectroscopic effects expected in a protein with independent pressure-denaturable domains shows that an apparent one-step change will be observed in all but extreme cases and that the volume changes calculated from the experimental data can grossly underestimate the change in volume upon denaturation of the whole protein.

• Research Organization: Univ. of Illinois, Urbana
• DOE Contract Number: E(11-1)-1198
• OSTI ID: 7231996
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 15:25; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
BINDING ENERGY
ENERGY
ENZYMES
FLUORESCENCE
GLYCOSYL HYDROLASES
HYDROLASES
LIGANDS
LUMINESCENCE
LYSOZYME
ORGANIC COMPOUNDS
PROTEIN DENATURATION
PROTEINS