Difference between revisions of "Part:BBa K1291571"

Latest revision as of 16:32, 17 October 2018

HORSERADISH PEROXIDASE TYPE C

The enzyme horseradish peroxidase (HRP), found in the roots of plant horseradish, is used extensively in biochemistry applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule. It is a metalloenzyme with many isoforms and the most studied type is C. This enzyme is primerly responsible for the degradation of industrial dyes such as Methylene Blue.

HRP Background, Usage and Biology

Horseradish peroxidase (HRP) is an enzyme used in molecular biology. It can be used in various biological methods, such as ELISA, Western Blotting, Southern Blotting, and EMSA [1]. HRP’s colorimetric and chemiluminescent properties make it a useful agent in many tests. HRP acts as a catalyst in redox reactions [3]. A luminol and H2O2 solution can release light for a very long time, but when HRP is added, it usually reaches it maximum light emission between 1 and 5 minutes [4]. This makes it a useful detection method. HRP can be used in more qualitative methods as well, since it induces changes in color when used with chromogenic assays. Commonly used substrates for this type of detection are 3,3',5,5'-tetramethylbenzidine (TMB), 2,2' -azino-di-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), and diaminobenzidine (DAB) [3,4]. HRP when added to TMB, for example, yields a 2-electron oxidation product which yields a blue color. HRP is also up and coming in the medical field. When combined with other agents, it is heavily reactive toward tumor cells in humans [2]. It also serves as a functional tracer. HRP can be injected into the bloodstream and then identified at different places in the pathway using the DAB/H2O2 reaction [4]. This makes HRP very useful for immunocytochemistry and electron microscopy [4].

References

[1] https://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=15552120 [2]https://chem.libretexts.org/Textbook_Maps/Biological_Chemistry/Catalysts/Case_Studies/Horseradish_Peroxidase [3] http://www.bio-rad.com/featured/en/hrp-substrate.html [4] https://www.sciencedirect.com/topics/neuroscience/horseradish-peroxidase [5] http://www.jbc.org/content/257/7/3669.full.pdf

HRP Characterization and Reaction Optimization

WLC-Milwaukee’s 2018 initial testing of HRP consisted of measuring the absorbance of HRP in hydrogen peroxide at 408 nm in the uv vis spectrometer. We added HRP in small aliquots to an excess of H2O2. This test yielded very small absorbances, but nonetheless showed an upward trend, which was expected. We expected the solution to turn red, but that did not happen, so we transitioned to other testing.

The next phase of testing involved chemiluminescence. A 10% hydrogen peroxide solution was made volumetrically and brought up to volume with pH 7.9 1X PBS (phosphate buffered saline). A ~500X luminol solution was made using 0.0145g of luminol and 1 mL of DMSO. 2 mL of the hydrogen peroxide solution with 20 uL of the luminol solution served as a control because it showed how much light was emitted from that without any HRP. Tests were done with 1, 2, and 3 uL of 1 ug/mL HRP added to a control solution. Each sample was run for 30 minutes, and two replicates were done of each sample, to end with 3 trials for each set of data. A consistent max light emission was observed between 5 and 10 minutes, making this the optimal measuring range for use in a test kit.

Here is an example of a fluorometer graph from Trial 1 of 1 uL of HRP

Averages were taken every 150 second interval. The next stage of testing involved using tetramethylbenzidine (TMB). TMB, used in conjunction with HRP, yields a colored product. When HRP is added, the solution turns blue, then green, and finally yellow. The blue product is measured at 652 nm and the yellow product is measured at 450 nm. 1 X TMB was diluted to a 5% solution with diH2O. The 100 ug/mL HRP solution was diluted to a 2% solution, and varying amounts of that were used. 600 mL of TMB was used for each trial with 2 uL, 5 uL, or 8 uL of HRP. The uv vis of the solution was taken every ~0.5 seconds. The graph below shows how an increase in HRP increases the speed of the reaction, allowing it to reach the maximum absorbance at 652 nm sooner.

Sequence and Features