Quantification of prothrombin in human plasma amplified by autocatalytic reaction.
By site directed mutagenesis, we have produced recombinant mutants of human and mouse prethrombin-2 which are able to convert themselves autocatalytically into α-thrombin. We also have created a new method to amplify the signal of bioanalytical assays based on the autocatalytic activation of these mutated proenzymes. The activation of the mutants by active α-thrombin triggers an autocatalytic reaction which leads to more active thrombin resulting in the amplification of the readout signal. Addition of mutated mouse prethrombin-2 into the conventional assay for prothrombin level in human plasma, employing ecarin and the fluorogenic substrate, resulted in improvement of the detection limit by 2 orders of magnitude.
CIC biomaGUNE, Parque Tecnológico de San Sebastián, Paseo Miramón 182, 20009, San Sebastián, Spain.
Ana
A
Virel
Laura
L
Saa
Valeri
V
Pavlov
D000818:Animals; D055162:Biocatalysis; D006801:Humans; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011516:Prothrombin / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D011994:Recombinant Proteins; D012235:Rhodamines; C049025:rhodamine 110; D011516:Prothrombin