Dissociation, denaturation, and deactivation of aldolase from rabbit muscle in the acid pH range have been investigated using sedimentation analysis, fluorescence, circular dichroism, and activity tests. Under comparable experimental conditions the pH-dependent profiles of deactivation and denaturation parallel the dissociation of the enzyme. In the range of dissociation at pH 4 -5 tetramers and monomers are in equilibrium. Intrinsic chromophores and far-ultraviolet circular dichroism suggest the transition to be a complex multistep process.At pH x 2.3 the enzyme is split into its fully inactive monomers which still contain some residual secondary structure. After reassociation under optimum conditions (0.2 M phosphate buffer pH 7.6, 1 mM EDTA, 0.1 mM dithiothreitol, 0 "C, enzyme concentration 0.4 -50 pg/ml) up to 95 % enzymic activity is recovered which belongs to a renatured tetrameric species indistinguishable from the native enzyme by all available biochemical and physicochemical criteria.There is well documented experimental evidence that oligomeric proteins, after deactivation and disruption of their three-dimensional structure under certain denaturing conditions, will spontaneously reassume an active conformation after removing the denaturant. Taking rabbit muscle aldolase as an example, reversible denaturation may be accomplished by a variety of denaturing and/or dissociating agents [l -171. As a result association has been considered to be a prerequisite of full enzymic activity [2,6,18]. On the other hand certain pieces of evidence point to active subunits [13,. This raises the question concerning the significance of enzyme subunit structure in cases where allosteric regulation does not seem to be of importance. The present renaturation experiments have been undertaken in order to elucidate the effect of refolding and quaternary structure formation on the catalytic properties of rabbit muscle aldolase. To compare the product of reactivation after acid dissociation with the native enzyme in its initial state a thorough analysis of the native, dissociated, and reassociated enzyme was performed.
MATERIALS AND METHODSFructose-l,6-bisphosphate aldolase from rabbit muscle, fructose 1,6-bisphosphate, NADH, and the mixture of glycerol-3-phosphate dehydrogenase and triose phosphate isomerase for the coupled aldolase assay were purchased from Boehringer (Mannheim). Bovine serum albumin was a product of Serva (Heidelberg). Trypsin obtained from Serva and Boehringer was treated with L-( 1-tosylamido-2-pheny1)-ethyl chloromethyl ketone (Serva) to inhibit the proteolytic activity of chymotrypsin [22]. Dithiothreitol was purchased from Calbiochem (Lucerne). All other reagents were A-grade substances from Merck (Darmstadt). Quartz bidistilled water was used throughout.Stock solutions of the enzyme (c = 4 mg/ml) were prepared by repeated dialysis at 4 "C against potassium phosphate buffer pH 7.6, 0.2 M, containing 1 mM EDTA and 0.1 mM dithiothreitol. Aldolase activity was determined in 0.05 M triethanolamine buffer pH...