[Multiple forms of horse pepsin].

The research article discusses the isolation and characterization of eight distinct forms of pepsin, an enzyme found in horse gastric juice. They were differentiated based on isoelectric point values, reflecting different structures and properties, yet showing similar functional activities. The study hypothesizes these forms might have originated from duplication of a common precursor gene and are in an early phase of divergence.

Methods of Isolation

• The eight forms of horse pepsin were isolated from horse gastric juice using ion-exchange and affinity chromatography. These techniques enable the separation of molecules based on their charge and binding affinity to specific groups respectively.
• Differentiation between these forms was achieved using their isoelectrofocusing points (pI). This method uses a pH gradient to separate proteins based on their isoelectric point, the pH at which the protein carries no net charge.

Characterization of Pepsin Forms

• The researchers analyzed the nature of these eight forms in terms of their molecular weights, amino acid composition, N-terminal sequence, and functional activity.
• A more in-depth study into the partial primary structure of the pepsin form with pI 2.3 was carried out.
• The partial sequence analysis shows that the forms with pI 1.8, 2.1, 2.3, and 2.6 have identical structures but different from the pepsin form with pI 3.2 by four substituents.

Functional Activity and Evolutionary Hypothesis

• Despite the structural differences, the functional activities of these different forms of horse pepsin were found to be quite similar.
• The researchers proposed that these multiple forms might have arisen from the duplication of the common precursor gene, indicating early stages of structural and functional divergence.

Comparison to Other Gastric Aspartyl Proteinases

• The study further analyzed their structural and functional properties in comparison with other isoenzymes of gastric aspartyl proteinases from mammals, such as pepsin A, gastricsin, or chymosin.
• The structural and functional features of these forms of horse pepsin were more related to pepsin A than to other isoenzymes studied.