Prolyl isomerases assay

A variant of the peptide-based assay avoids the coupling with isomer-specific proteolysis (Janowski et al., 1997). The absorbance at 330 nm of succinyl-Ala-Phe-Pro-Phe-4-nitroanilide decreases slightly when the Phe and the nitroanilide moieties rearrange on the cis —trans isomerization at the Phe-Pro bond of the peptide. Because of the small change in signal, the sensitivity of the assay is limited, but it is the method of choice for assaying prolyl isomerases sensitive to proteases. 

We will use the term PPI when the enzymatic activity as prolyl isomerase is concerned, and the terms cyclophilin and FKBP to discriminate the two known families of prolyl isomerases that are inhibited by CsA and by FK506, respectively. Cyclophilin and FKBP differ strongly in sequence specificity. The PPI activity of cyclophilin shows only a small dependence on the chemical nature of the amino acid Xaa that precedes proline in the assay peptide. The activity of FKBP, however, varies by three orders of magnitude when the same set of peptides is used in the PPI assays (Harrison and Stein, 1990 see Stein, this volume). 

A new, continuous assay for prolyl isomerases was developed by Garda-Echeverrla et al (1992). 

The first enzyme to catalyze prolyl isomerization was identified by Fischer and colleagues (1984). This discovery was possible because they developed an ingenious assay for prolyl isomerases based on the conformational specificity of chymotrypsin. This protease cleaves a chro-mogenic reporter group from a tetrapeptide (such as succinyl-Ala-Ala-Pro-Phe-4-nitroanilide) only when the Ala—Pro bond of this peptide is in the trans conformation. In aqueous solution the assay peptide exists as a 90 10 mixture of molecules with the Ala—Pro bond in trans and cis, respectively. Therefore, in the presence of a high concentration of chymotrypsin, 90% of the peptide molecules are cleaved within the dead time of manual mixing. Hydrolysis of the remaining 10% is slow because it is limited in rate by the cis —trans isomerization of the Ala—Pro bond. Acceleration of this reaction serves as a sensitive probe for prolyl isomerase activities. 

Recently, Kiillertz and Fischer developed a new prolyl isomerase assay for high-troughput screening of up to 20,000 samples per day. This assay employs a disulfide-bonded cis peptide with internally quenched fluorescence. Cis/trans isomerization is initiated by rapid reductive ring opening and followed by the increase in fluorescence (G. Kiillertz and G. Fischer, unpublished). 

Whereas peptidyl prolyl cis-trans isomerases constitute a well-characterized enzyme class comprising well over 1000 members with small sequence variations in the proteins of different species, the discovery of secondary amide peptide bond cis-trans isomerases (APIases) had to await the development of suitable enzyme assays. Fortunately, spectral differences in the UV region between cis and trans isomers of dipeptides could be exploited to identify and quantify isomerization rate-enhancing factors in biological material [149]. 

Mofron. J.L. Kuzmic. P. Kishore, V. Colonbonilla. E. Rich. D.H. Determination of kinetic constants for peptidyl prolyl cis trails isomerases by an improved spectrophoto-metric assay. Biochemistry 1991. 30, 6127-6134. 

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