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Isolation and characterization of human LH isoforms
P G Stanton1, G Pozvek, P G Burgon
1Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
The Journal of Endocrinology
|September 1, 1993
Summary
A new mild purification method successfully isolated 17 highly pure human luteinizing hormone (hLH) isoforms from pituitary extracts. These isoforms are suitable for further characterization and research.
Area of Science:
- Biochemistry
- Endocrinology
- Protein Chemistry
Background:
- Human luteinizing hormone (hLH) exists in multiple isoforms with varying biological activities.
- Characterizing these isoforms is crucial for understanding reproductive endocrinology.
- Previous purification methods were often harsh, potentially altering hLH structure and activity.
Purpose of the Study:
- To develop a mild purification procedure for isolating intact human LH (hLH) isoforms.
- To characterize the purity, molecular mass, and subunit composition of isolated hLH isoforms.
- To assess the suitability of the isolated isoforms for further biological and physicochemical studies.
Main Methods:
- Preparative isoelectric focusing
- High-performance ion-exchange chromatography
- Immobilized metal-affinity chromatography
- LH radioreceptor assay
- SDS-PAGE
- Amino acid analysis
- N-terminal sequencing
Main Results:
- Thirty-nine hLH isoforms were separated; 17 were purified to >90% purity.
- Specific activities ranged from 1980 to 38,650 IU/mg protein.
- SDS-PAGE revealed alpha and beta subunits under reducing conditions.
- Amino acid composition matched published cDNA sequences.
- N-terminal sequencing confirmed intact alpha and beta subunits.
Conclusions:
- A mild, high-recovery purification procedure for intact hLH isoforms was established.
- Seventeen highly purified hLH isoforms were isolated, suitable for further research.
- The isolated isoforms are free of contaminating proteins but may contain related species.