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A simple rapid purification scheme for hydroxymethylbilane synthase from human erythrocytes
1Department of Biochemistry, Trinity College, Dublin, Ireland.
The Biochemical Journal
|April 1, 1988
Summary
Hydroxymethylbilane synthase was purified from human red blood cells using heat treatment and chromatography. The enzyme
Area of Science:
- Biochemistry
- Enzymology
Background:
- Hydroxymethylbilane synthase is a key enzyme in heme biosynthesis.
- Understanding its properties is crucial for metabolic studies.
Purpose of the Study:
- To purify hydroxymethylbilane synthase from human erythrocytes.
- To characterize the kinetic and physical properties of the purified enzyme.
Main Methods:
- Heat treatment (80°C) with proteinase inhibitors.
- DEAE-cellulose and Cibacron Blue F3G-A-Sepharose chromatography.
- Sephadex G-75 gel filtration.
Main Results:
- Achieved 47,000-fold purification to >95% homogeneity.
- Enzyme exhibited hyperbolic substrate dependence and non-linear product formation.
- Determined monomeric molecular weight (44,000-45,000 Da) and sharp pH optimum for Vmax.
- Folates did not affect enzyme activity.
Conclusions:
- A rapid and effective purification protocol for human hydroxymethylbilane synthase was established.
- The purified enzyme shares properties with orthologs from other species.
- Folates are not cofactors for this enzyme in vitro.