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Biochemical background

Chemical reactions are permanently taking place in the organism, which without catalyzing enzymes could only take place very slowly (or not at all) at neutral pH and body temperature. Enzymes that catalyze degradation of proteins are called proteases. During proteolysis, proteins are cleaved to obtain peptides or free amino acids. These processes are highly specific; only peptide complexes within a certain sequence of amino acids are hydrolyzed.

Around 1970 researchers started to construct synthetic substrates, in order to measure enzyme activity. By reconstructing relevant parts of the target protein amino acid sequence, the synthetic substrates obtained a similar selectivity as the natural substrates of a specific enzyme. Synthetic substrates consist of a sequence of 3-4 amino acids with a chromogen group or chromophore (Greek: chroma = colour) at the terminal. The substrate is cleaved by the enzyme to be determined and releases the chromophore which can be photometrically measured. A chromophore regularly used with these chromogenic substrates is p-nitroanilin (pNA), that releases a yellow colour, which can be measured at 405 nm.

Of great importance for the correct application of a chromogenic substrate is the specificity and selectivity with regard to the various enzymes. Specificity is a characteristic of the enzyme and describes limitations in the cleavage of different substrates: according to this, a completely specific enzyme only cleaves a certain defined substrate. In general the specificity of a proteolytic enzyme is not very high. The majority of these enzymes cleave a variety of substrates, albeit at different speeds. The selectivity as a characteristic of the substrate is a measure for how well this substrate is bound to and cleaved by various enzymes.

Sensitivity and selectivity

The table shows examples of substrate turnover expressed as ΔA/min of certain substrates with different proteases [4 x 10-9 mol/l]. Orangefaced numbers indicate the best selectivity of a substrate for a specific enzyme. ND - Not determined 

Literature

1. Friberger P. Chromogenic peptide substrates. Their use for the assay of factors in the fibrinolytic and plasma kallikrein-kinin systems. Scand J Clin Lab Invest Suppl. 162, 1982.
2. Gallimore MJ, Friberger P. Chromogenic peptide substrate assays and their clinical applications. Blood Rev. 5, 117-127, 1991.
3. Hemker HC. Handbook of synthetic substrates for the coagulation and fibrinolytic system. Martinus Nijhoff Publ., The Hague, 1983.
4. Friberger P. Synthetic Peptide Substrate Assays in Coagulation and Fibrinolysis and Their Application on Automates. Sem Thromb Haemost 9, 281-300, 1983.
5. Trobisch H, Klasing KH. Die Bedeutung chromogener Peptidsubstrate für die Gerinnungsdiagnostik. Med Labor 33, 183-191, 1980.
6. Bruhn HD, Broers H. Chromogene Substrate in der Blutgerinnungsdiagnostik und in der Überwachung der Antikoagulantien- und Fibrinolysetherapie. Internist Prax 21, 623-629, 1981.

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