cytochrome-c-t and 3-3--5-5--tetramethylbenzidine

The development of simple and sensitive methods for protease sensing plays important roles in clinical diagnostics and drug development. Here a simple, rapid, label-free, and sensitive trypsin colorimetric sensor was developed by employing cytochrome c (cyt c) as an enzyme substrate and 3,3´,5,5´-tetramethylbenzidine (TMB) as a chromogenic reagent. It was found that cyt c hardly catalyzes H2O2-mediated TMB oxidation to produce a blue solution. But the hydrolysate of cyt c by trypsin displays an intense catalytic effect on the aforementioned reaction, resulting in the formation of a blue solution immediately. The detection process allows visually perceiving as low as 50 ng/mL trypsin with the naked eyes. With the aid of a spectrophotometer, the absorbance at 652 nm was proportional to the concentration of trypsin in the range from 5.0 ng/mL to 2.0 μg/mL with a detection limit of 4.5 ng/mL. The sensor showed better precision with relative standard deviation of 2.5% and 1.7% for eleven repetitive measurements of 50.0 ng/mL and 1.0 μg/mL trypsin solution, respectively. The procedure has been successfully applied to the determination of trypsin in human urines and for inhibitor screening, demonstrating its potential application in clinic diagnosis and drug development.

Topics: Benzidines; Biosensing Techniques; Catalysis; Colorimetry; Cytochromes c; Humans; Limit of Detection; Oxidation-Reduction; Trypsin

Binding of heme to the amyloid peptides Aβ40/42 is thought to be an initial step in the development of symptoms in the early stages of Alzheimer's disease by enhancing the intrinsic peroxidatic activity of heme. We found considerably higher acceleration of the reaction for the physiologically relevant neurotransmitters dopamine and serotonin than reported earlier for the artificial substrate 3,3',5,5'-tetramethylbenzidine (TMB). Thus, the binding of hemin to Aβ peptides might play an even more crucial role in the early stages of Alzheimer's disease than deduced from these earlier results. To mimic complex formation, a new surface architecture has been developed: The interaction between the truncated amyloid peptide Aβ1-16 and hemin immobilized on an aminohexanethiol spacer on a gold electrode has been analyzed by cyclic voltammetry. The resulting complex has a redox pair with a 25 mV more cathodic formal potential than hemin alone.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Angiotensins; Benzidines; Biosensing Techniques; Cytochromes c; Dopamine; Electrochemistry; Electrodes; Gold; Heme; Hemin; Humans; Neurotransmitter Agents; Oxidation-Reduction; Peroxidases; Protein Binding; Serotonin; Surface Properties

A variety of luminol-based substrates and either an autoradiographic film or a charge-coupled device (CCD) imaging system were used for chemiluminescence detection of c-type cytochromes. The Pierce Femto peroxidase substrate was at least 10 times more sensitive when using film than the highly cited 3,3('),5,5(')-tetramethylbenzidine (benzidine derivative) staining method and 50 times more sensitive when using a CCD imaging system. Film or CCD imaging result in highly sensitive and quantitative signals. The quantitative detection of c-type cytochromes from single colonies or from less than 1ml of a bacterial culture is possible.

Topics: Bacterial Proteins; Benzidines; Chemistry Techniques, Analytical; Chromogenic Compounds; Cytochromes c; Electrophoresis, Polyacrylamide Gel; Heme; Heterocyclic Compounds; Luminescent Measurements; Peroxidases; Rhodobacter capsulatus; Sensitivity and Specificity; Staining and Labeling