Primary structure of 3-phosphoglycerate kinase from horse muscle. I. Purification of cyanogen bromide peptides and amino acid sequence of peptide CB5 (104 residues).

This research article focuses on the extraction of 3-Phosphoglycerate kinase enzyme from horse muscle and the detailed sequencing of its largest peptide, CB5, using a combination of automated and manual strategies.

Isolation of 3-Phosphoglycerate Kinase

• The experiment started with the isolation of 3-Phosphoglycerate kinase, an important enzyme assisting in the process of glycolysis, from horse muscle.
• After successful isolation, the enzyme subjected to the action of cyanogen bromide, a potent chemical compound traditionally used for protein sequencing. It’s used to cleave proteins at methionine residues, resulting in smaller peptides that are easier to analyze.

Separation of Peptides

• The smaller peptides resulting from cyanogen bromide action were then separated using a combination of gel filtration and either ion exchange chromatography on phosphocellulose in 6 M urea or high voltage paper electrophoresis.
• Gel filtration is a type of chromatography that separates proteins based on their size, whereas ion exchange chromatography separates them based on their charge.
• High voltage paper electrophoresis is another method used to separate macromolecules like proteins and nucleic acids based on their size-to-charge ratio.

Sequence Analysis of Peptide CB5

• The resulting peptides were analyzed, with special attention given to the largest peptide, CB5.
• The sequence of CB5 was determined using a combination of automated and manual Edman degradation on the intact peptide, and derivatives obtained from proteolytic digestion.
• Edman degradation is a series of chemical reactions that sequentially remove the N-terminal amino acid of a peptide, allowing researchers to sequence the peptide one amino acid at a time.
• In addition, the bond between Asp109 and Pro110 was cleaved to facilitate further sequence analysis of CB5.

Amino Acid Sequence of Remaining Peptides and Full Enzyme

• Apart from CB5, the sequence analysis of the remaining 13 cyanogen bromide fragments was also carried out, whose detailed results are documented in the next paper of this series.
• The ultimate goal of the research was to determine the complete sequence of the 3-Phosphoglycerate kinase enzyme, paving the way for better understanding of its structure and function.