Crystallization and properties of creatine kinase from equine skeletal muscle.

The study focuses on obtaining and examining a crystalline form of creatine kinase, an enzyme, from horse skeletal muscle.

Research Process

• The researchers managed to secure a crystalline form of the enzyme creatine kinase from the skeletal muscle of a horse. The purity of this enzyme was ensured through ultracentrifugation and disc electrophoresis on polyacrylamide gel.
• Ultracentrifugation is a technique that utilizes high-speed centrifugal force to separate molecules based on their size and shape. Similarly, disc electrophoresis on polyacrylamide gel is a method for separating proteins on the basis of their size.

Enzyme Activity and Purification

• The resultant crystalline form of the enzyme showed a specific activity of 110 units per milligram of protein, implying that the purification process had increased its effectiveness by roughly 14 times compared to that of the crude equine skeletal muscle extract.
• The term ‘specific activity’ is used to indicate the activity level per unit of the total proteins present in an enzyme. This measure helps in evaluating the purity and activity of enzymes.

Molecular Weight and Amino Acid Composition

• Using the conventional low-speed sedimentation equilibrium method, which is a method used to determine the molecular weight of a substance by measuring its rate of sedimentation in specific conditions, the researchers estimated the creatine kinase’s molecular weight at 84,600.
• The molecule’s sedimentation coefficient was determined to be 5.32S. A sedimentation coefficient represents a particle’s tendency to settle under the influence of gravity or centrifugal force. Higher values indicate faster settling.
• An amino acid analysis revealed eight cysteine residues, two of which were identified as essential for enzymatic activity. Cysteine is an amino acid that plays significant roles in proteins, affecting their structure and functions. Therefore, the presence of cysteine residues can greatly influence the behavior of an enzyme.