Electrochemical assay of protease activities based on polycation/polyanion complex as substrate and polyion sensitive membrane electrode detection.
A novel electrochemical method to detect protease activities is demonstrated. The assay is based on the use of a macromolecular polycation/polyanion substrate; specifically, a complex of the arginine-rich peptide protamine and pentosan polysulfate (PPS), a highly sulfated polysaccharide. As the protease of interest cleaves the protamine within the complex into smaller fragments, free PPS is generated and detected potentiometrically via a polyanion sensitive membrane electrode. Thus, the rate of free PPS generation is proportional to the activity of the protease in the assay solution. The effect of the substrate concentration is examined, as is the influence of the protamine/PPS stoichiometry on the assay performance. Using the optimized composition and concentration of the complex, the determination of trypsin at levels down to 5 U/ml and plasmin at levels approaching 0.002 U/ml can be achieved in a 10 min period. The prospects of further adapting this scheme to determine clot-busting plasminogen activators (e.g. streptokinase, tissue plasminogen activator, etc.) in samples as complex in whole blood are discussed.
Abd-Rabboh
Hisham S M
HS
Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109-1055, USA.
Nevins
Stacey A
SA
Dürüst
Nedime
N
Meyerhoff
Mark E
ME
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D004566:Electrodes; D010450:Endopeptidases / Q000032:analysis* / Q000737:chemistry; D004789:Enzyme Activation; D005341:Fibrinolysin / Q000032:analysis; D046911:Macromolecular Substances; D008567:Membranes, Artificial; D010426:Pentosan Sulfuric Polyester / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry; D000071228:Polyelectrolytes; D011108:Polymers / Q000737:chemistry; D011479:Protamines / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry; D013379:Substrate Specificity; D014357:Trypsin
D020099:Coated Materials, Biocompatible; D010450:Endopeptidases; D005341:Fibrinolysin; D046911:Macromolecular Substances; D008567:Membranes, Artificial; D010426:Pentosan Sulfuric Polyester; D011073:Polyamines; D000071228:Polyelectrolytes; D011108:Polymers; D011479:Protamines; D012996:Solutions; D014357:Trypsin