Affinity chromatographic purification of horse muscle acylphosphatase: evidence of the existence of multiple molecular forms.

The research focused on purifying the enzyme acylphosphatase from horse muscle through a gentle process using affinity and ion-exchange chromatography, showing the existence of three molecular forms of the enzyme. The procedure, which had an overall yield of about 60% of activity, suggested that each enzyme form is tightly linked to a specific antibody and has similar kinetic parameters when interacting with a certain substrate.

Methodology

• Acylphosphatase from horse muscles was purified in this research by using a new process involving affinity chromatography and followed by ion-exchange chromatography. Affinity chromatography involved separating proteins based on a specific interaction between an antigen (i.e. the protein in question) and an antibody, while ion-exchange chromatography separated proteins according to their charge.
• This procedure was considerably more gentle than previous methods (thus ensuring the preservation of the molecular forms) and had an overall yield of about 60% of activity.

Findings

• The study resulted in the isolation of three molecular forms, all with acylphosphatase activity. Each of these enzymatic forms was found to bind tightly to Sepharose 4B-linked anti-horse muscle acylphosphatase antibodies.
• Out of these three molecular forms, two (Ho1 and Ho3) were found in larger amounts. Ho1 matched the enzyme previously purified using the old procedure, consisting of a mixed disulfide between a primary chain of 98 amino acid residues and Glutathione. The enzyme Ho2, however, differed from Ho1 only in the chemical nature of the S-S molecule bound to the lone cysteine (an amino acid) at position 21 of the main chain. Ho3 was identified as an S-S dimer of the main polypeptide chain.
• Each of the molecular forms, Ho1, Ho2, and Ho3, exhibited very similar kinetic parameters when benzoylphosphate was used as the substrate, suggesting a similar rate of enzymatic reaction.

Implications

• The discovery of the multiple molecular forms of acylphosphatase from horse muscles provides new insights into its structure and potential functional diversity and opens up new opportunities for understanding the enzyme’s functions and interactions in the horse muscles.
• Additionally, the 60% activation yield and the gentle nature of the process prove it to be a more effective method of isolating and studying these molecular forms.