Reconstitution of Lactic Dehydrogenase after Acid Dissociation

Abstract

Reconstitution of lactic dehydrogenase from pig muscle after dissociation at acidic pH produces native tetramers and inactive aggregates, both of which have been previously described with respect to their structural properties and the kinetics of their formation. The ratio of the two fractions is determined by the relative rates of reassociation and aggregation [G. ZettlmeiBI, R. Rudolph and R. Jaenicke (1979) Biochemistry 18, 5567-55711. The present study is concerned with the influence of residual structural elements within the acid-denatured monomers on the process of reconstitution.Long incubation at low pH leads to a marked decrease of the rate and yield of reactivation, caused by conformational changes within the partially unfolded monomers. These slow rearrangements are reflected by slight changes of the far-ultraviolet circular dichroism of the acid-dissociated monomers, indicating a decrease in helix content from 30 observed immediately after the transfer to low pH, to 25 % after completion of the acid transition.The unchanged near-ultraviolet circular dichroism and fluorescence properties prove local structural alterations to be of lesser importance compared to the changes of the residual backbone folding. Varying the experimental conditions during denaturation proves the slow decrease in the rate and yield of reactivation, as well as the changes in the far-ultraviolet circular dichroism to be enhanced with incubation time and temperature (0 -30 <C). The firstorder rate constants of the decrease in the reactivation yield after incubation in 1 M glycine/H,PO, pH 2.3, 0.1 M sodium phosphate p H 2.3, and 1 M H,PO, were found to be in the same time range as the respective changes in the far-ultraviolet circular dichroism.Apart from the similarity of the rate constants, the activation energy for both the acid induced changes of the yield of reactivation and the far-ultraviolet circular dichroism is found to be identical ( z 80 kJ/mol).The effects of low pH on the reconstitution of lactic dehydrogenase can be eliminated by complete disruption of the structure of the polypeptide chain, e.g. by adding 6 M guanidine . HCI. This proves unambiguously that the above-mentioned structural rearrangements at acid p H are responsible for the observed decrease of the rate and yield of reactivation.

The unfolding and refolding of pig heart fumarase

“…Slow structural transitions at intermediate concentrations of denaturant (Fig. 3) have been observed in other systems (see, e.g., Zettlmeissl et al, 1981;Tsou, 1986). The nature of these transitions remains to be explored, although clearly relatively small structual rearrangements are involved.…”

“…The nature of these transitions remains to be explored, although clearly relatively small structual rearrangements are involved. In some cases cis=trans isomerization of proline peptide bonds may also occur (Zettlmeissl et al, 1981 ;Brandts & Lim, 1986). Presumably in the presence ofthese intermediate concentrations ofdenaturing agents a number of conformational states are kinetically accessible to the enzyme.…”

Rate-determining folding and association reactions on reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride

“…Lactic dehydrogenase (EC 1.1.1.27; lactate dehydrogenase) from pig skeletal muscle was obtained from Boehringer (Mannheim), and glutaraldehyde [purissimum, 25% or 50% (w/v) aqueous solution] was from Fluka (Basel). All other reagents were the same as described elsewhere (Zettlmeissl et al, 1981).…”

“…The preparation of stock solutions of the enzyme, concentration determinations, and enzymatic tests were performed as described previously (Zettlmeissl et al, 1981).…”

Protein refolding is improved by adding nonionic polyethylene glycol monooleyl ethers with various polyethylene glycol lengths

“…Denaturation and refolding experiments were carried out according to previously reported procedures , 26] with modification in the protein concentration, which was almost ten times higher for more severe conditions for refolding. The native LDH concentration was determined by the absorbance at 280 nm using an extinction coefficient ( ε 280 ) of 1.4 mL/mg/cm . Absorbance was measured with a UV‐Vis spectrophotometer model ND‐1000 (NanoDrop, US).…”