An Improved Purification Protocol for Chicken Muscle Beta-enolase
Institution
Eastern Kentucky University
Faculty Advisor/ Mentor
William W. Farrar
Abstract
Enolase is a Mg2+-dependent metalloenzyme in the glycolytic pathway that catalyzes the dehydration of 2-Phosphglyceric acid (2-PGA) to Phospho(enol)pyruvate (PEP). In vertebrate organisms there are three genes, designated as alpha, beta, and gamma, that code for enolase subunits of the same designation. Since vertebrate enoalses are dimers, there are six possible molecular forms (isoforms). The enolase isoform in vertebrate muscle is the beta-enolase. Chicken muscle beta-enolase has previously been purified by a research group in Japan, but the protocol used nine procedures involving six column steps, and a yield of 5.5%. Our protocol uses three procedures involving one column step. The purification scheme is fast, requiring only 1.5 days to complete, and results in homogeneous enolase having a specific activity of 121 Units mg protein. The isoelectric point (pI) was determined to be 7.3, close to the theoretical pI of 7.2. The X-ray crystallographic structure of enolase is known only for enolases from yeast, lobster, and E. coli. Thus, no vertebrate enolase structure is presently known. Therefore we have initiated crystallization trials in an effort to obtain suitable crystals for structural studies at the Kentucky Center for Structural Biology.
An Improved Purification Protocol for Chicken Muscle Beta-enolase
Enolase is a Mg2+-dependent metalloenzyme in the glycolytic pathway that catalyzes the dehydration of 2-Phosphglyceric acid (2-PGA) to Phospho(enol)pyruvate (PEP). In vertebrate organisms there are three genes, designated as alpha, beta, and gamma, that code for enolase subunits of the same designation. Since vertebrate enoalses are dimers, there are six possible molecular forms (isoforms). The enolase isoform in vertebrate muscle is the beta-enolase. Chicken muscle beta-enolase has previously been purified by a research group in Japan, but the protocol used nine procedures involving six column steps, and a yield of 5.5%. Our protocol uses three procedures involving one column step. The purification scheme is fast, requiring only 1.5 days to complete, and results in homogeneous enolase having a specific activity of 121 Units mg protein. The isoelectric point (pI) was determined to be 7.3, close to the theoretical pI of 7.2. The X-ray crystallographic structure of enolase is known only for enolases from yeast, lobster, and E. coli. Thus, no vertebrate enolase structure is presently known. Therefore we have initiated crystallization trials in an effort to obtain suitable crystals for structural studies at the Kentucky Center for Structural Biology.