Research Article 5: Partial Loss of Activity of Individual Molecules of Aged β-galactosidase

Abstract

INTRODUCTION
Previous studies of bacterial β-galactosidase at the single molecule level have shown that thermal denaturation of the enzyme resulted in an all-or-none inactivation (Rotman, 1961). In contrast to this observation, it is shown here that enzyme inactivation caused by prolonged storage results in a population of molecules with intermediate levels of activity ranging from less than five to one hundred percent of that present in the native β-galactosidase molecule.

The significance of these findings is discussed in terms of the free energy of newly synthesized versus aged enzyme.

MATERIALS AND METHODS
Buffers, chemicals, and growth medium. Buffer B contained 10 mM tris (hydroxymethylaminomethane), 10 mM MgCl₂, 100 mM NaCl and 50 mM 2-mercaptoethanol. The latter was added to the buffer prior to its use. The pH of the buffer was adjusted to 7.5 (23°C) with acetic acid. Buffer P was prepared by adjusting a 20 mM solution of Na₂HPO₄ to pH 7.2 (23°C) with a 20 mM solution of NaH₂PO₄. Solutions with urea (ultra pure grade, Mann Research Lab., Inc.) were prepared with buffer B.

5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside was obtained from Cyclo Corp. Fluorescein-di-(β-D-galactopyranoside) and o-nitrophenyl-β-D-galactopyranoside (ONPG) were purchased from Mann Research Laboratories, Inc. The former was purified by descending chrornatography on S & S orange paper using 1-pentanol:1-propanol:water = 40:11:15 as solvent (Rotman, Zderic, and Edelstein, 1963).

Bacterial cultures were grown in minimal medium (Davis, 1949) containing 0.4% sodium lactate (Fisher Co.).

Purification of β-galactosidase (β-D-galactoside galactohydrolase 3.2.1.23). The procedure described by Craven, Steers, and Anfinsen (1965) was used to...