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Chemically crosslinked lactate dehydrogenase: stability and reconstitution after glutaraldehyde fixation
1Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Federal Republic of Germany.
Biotechnology and Applied Biochemistry
|October 1, 1987
Summary
Crosslinking pig heart lactate dehydrogenase (LDH) with glutaraldehyde stabilizes its structure, enhancing thermal and chemical stability while retaining significant enzyme activity. This crosslinking method impacts reconstitution after denaturation.
Area of Science:
- Biochemistry
- Enzyme Stabilization
- Protein Chemistry
Background:
- Lactate dehydrogenase (LDH) is a crucial enzyme involved in cellular metabolism.
- Maintaining the native quaternary structure of enzymes is vital for their function and stability.
- Glutaraldehyde is a common crosslinking agent used in protein modification.
Purpose of the Study:
- To stabilize pig heart lactate dehydrogenase (LDH) in its tetrameric form using glutaraldehyde crosslinking.
- To investigate the impact of crosslinking on the enzyme's stability, activity, and denaturation/renaturation properties.
- To characterize the structural and functional consequences of preserving the native quaternary structure.
Main Methods:
- Crosslinking of pig heart LDH with glutaraldehyde.
- Enzyme activity assays to determine retained function.
- Spectroscopic methods to assess spectral properties.
- Differential scanning calorimetry and guanidine hydrochloride denaturation studies to evaluate stability.
- Kinetic analysis of thermal denaturation and acid denaturation/reactivation.
Main Results:
- Glutaraldehyde crosslinking yielded a product with 82% tetramers, retaining up to 60% of original LDH activity.
- Crosslinked LDH exhibited enhanced stability, with heat and guanidine deactivation transitions shifted to higher temperatures (65°C) and denaturant concentrations (1.0 M CG.HCl).
- Deactivation and denaturation were decoupled; fluorescence emission transitions occurred at 3.5 M CG.HCl, indicating preserved cooperativity, while unfolding kinetics revealed distinct rates for partially and fully crosslinked species.
Conclusions:
- Crosslinking pig heart LDH with glutaraldehyde effectively stabilizes its tetrameric structure, significantly enhancing its resistance to thermal and chemical denaturation.
- While crosslinking improves stability, it alters the denaturation and renaturation pathways, with complete randomization in guanidinium chloride hindering reconstitution.
- The study demonstrates that fixation of the native quaternary structure is a viable strategy for improving enzyme stability, though it influences refolding mechanisms.