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Effects of urea and guanidine hydrochloride on the activity and dynamical structure of equine liver alcohol dehydrogenase.
Abstract: The inactivation of equine liver alcohol dehydrogenase by guanidine hydrochloride and urea has been studied by monitoring the intrinsic tryptophan fluorescence and phosphorescence emission. The use of triplet-state lifetimes to probe the flexibility of protein structure at the site of tryptophan-314 reveals a distinct behavior between the two denaturants. At predenaturational concentrations, the loss of enzyme activity in guanidine hydrochloride is associated with a loosening of intramolecular interactions resulting in a greater fluidity of the interior region of the macromolecule. In contrast, the interaction with urea, even at high concentrations, does not alter the dynamics of the native conformation. Enzyme activity is irreversibly lost as a result of a drastic unfolding of the macromolecule which occurs in a highly cooperative two-stage process.
Publication Date: 1986-05-06 PubMed ID: 2941076DOI: 10.1021/bi00357a027Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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The research examines how urea and guanidine hydrochloride affect the activity and structure of equine liver alcohol dehydrogenase, a liver enzyme in horses. The study reveals differential effects of these two substances, with guanidine hydrochloride affecting the enzyme’s structural flexibility and urea triggering a significant unfolding of the macromolecule, leading to irreversible loss of enzyme activity.
Understanding the Methods and Materials
- The research focuses on equine liver alcohol dehydrogenase, an enzyme derived from horse liver.
- Two substances were studied for their interaction with this enzyme: guanidine hydrochloride and urea.
- The means to track these interactions was intrinsic tryptophan fluorescence and phosphorescence emission. Tryptophan-314, a particular site within the enzyme, was specifically targeted for observation.
- The dynamic structure of the enzyme and its activity levels were scrutinized both before and after the application of these substances.
Findings on Guanidine Hydrochloride’s Effects
- Under guanidine hydrochloride’s influence, the enzyme showed a loss of activity.
- At predenaturational concentrations (concentrations before any potential structural denaturation or modification), this loss was connected with a reduction of intramolecular interactions. These interactions maintain the structure of the enzyme.
- This reduction enabled more fluidity within the macromolecule’s internal region, implying that guanidine hydrochloride causes the enzyme’s structure to become more ‘loose’ or flexible.
Findings on Urea’s Effects
- Contrarily, the interaction of urea with the enzyme, even at high concentrations, did not disrupt the dynamics of the enzyme’s native (original) conformation.
- However, urea caused a dramatic and irreversible loss of enzyme activity, which was attributed to significant unfolding of the macromolecule. This unfolding disrupts the enzyme’s structure and, consequently, its functionality.
- This unfolding occurred in two main stages and was highly cooperative, implying a complex unfolding process involving various parts of the molecule.
Significance and Implications of the Study
- The study essentially provides insight into the differential impacts of urea and guanidine hydrochloride on the structure and activity level of the equine liver alcohol dehydrogenase enzyme.
- This understanding may help in further studies aimed at exploring the denaturational effects of various substances on enzymes, potentially contributing to the development of treatments to counteract these effects.
Cite This Article
APA
Strambini GB, Gonnelli M.
(1986).
Effects of urea and guanidine hydrochloride on the activity and dynamical structure of equine liver alcohol dehydrogenase.
Biochemistry, 25(9), 2471-2476.
https://doi.org/10.1021/bi00357a027 Publication
Researcher Affiliations
MeSH Terms
- Alcohol Dehydrogenase
- Alcohol Oxidoreductases / antagonists & inhibitors
- Animals
- Circular Dichroism
- Guanidine
- Guanidines / pharmacology
- Horses
- Kinetics
- Liver / enzymology
- Luminescent Measurements
- Oxidation-Reduction
- Protein Conformation
- Spectrometry, Fluorescence
- Tryptophan
- Urea / pharmacology
- Viscosity
Citations
This article has been cited 9 times.- Shanmuganatham KK, Wallace RS, Ting-I Lee A, Plapp BV. Contribution of buried distal amino acid residues in horse liver alcohol dehydrogenase to structure and catalysis. Protein Sci 2018 Mar;27(3):750-768.
- Gonnelli M, Puntoni A, Strambini GB. Tryptophan phosphorescence of ribonuclease T1 as a probe of protein flexibility. J Fluoresc 1992 Sep;2(3):157-65.
- Cioni P, Bramanti E, Strambini GB. Effects of sucrose on the internal dynamics of azurin. Biophys J 2005 Jun;88(6):4213-22.
- Strambini GB, Gabellieri E, Gonnelli M, Rahuel-Clermont S, Branlant G. Tyrosine quenching of tryptophan phosphorescence in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. Biophys J 1998 Jun;74(6):3165-72.
- White MF, Fothergill-Gilmore LA, Kelly SM, Price NC. Substitution of His-181 by alanine in yeast phosphoglycerate mutase leads to cofactor-induced dissociation of the tetrameric structure. Biochem J 1993 Apr 15;291 ( Pt 2)(Pt 2):479-83.
- Fröhlich O, Jones SC. Denaturation of a membrane transport protein by urea: the erythrocyte anion exchanger. J Membr Biol 1987;98(1):33-42.
- Strambini GB. Quenching of alkaline phosphatase phosphorescence by O2 and NO. Evidence for inflexible regions of protein structure. Biophys J 1987 Jul;52(1):23-8.
- Johnson CM, Price NC. Denaturation and renaturation of the monomeric phosphoglycerate mutase from Schizosaccharomyces pombe. Biochem J 1987 Jul 15;245(2):525-30.
- Pearce AM, Irons LI, Robinson A, Seabrook RN. Effects of guanidinium hydrochloride on the structure and immunological properties of Bordetella pertussis fimbriae. Biochem J 1992 May 1;283 ( Pt 3)(Pt 3):823-8.
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