1: To create a reagent for your Western Blot, you purified an enzyme, horsereddi

Question

1: To create a reagent for your Western Blot, you purified an enzyme, horsereddish peroxidase from a horseradish (Armoracia rusticana) plant. Then, you run an enzyme kinetics assay on four increasingly pure fractions of your enzyme. For this kinetics assay, you used TMB, (3,3',5,5'-tetramethybenzidine) substrate that, after acidification, produced final diimine product adsorbing light at 450 nm with the molar absorptivity of E450nm=59,000 cm-1 M-1. You obtained V0 data using A450nm, and now need to calculate V0 in m/L. You used 100nM of TMB in a 96-well plate and the assay volume 200L that has a pathlength of 0.585cm. Using the Beer law, A=ebc, calculate initial velocity of each fraction in M/min.

2:You were able to obtain pure HRP enzyme and calibrate you kinetics assay. HRP concentrations that you obtained according to your calibration are listed in the table below (use V0 calculated in 1)

Based on the data in the table above, create a Lineweaver–Burk plot and estimate Vmax and Km of HRP from it. 1/V0 = Km/Vmax[S] + 1/Vmax Lineweaver–Burk equation *Use a separate sheet of paper to for your answer.

please please please help been going at this question for days.....

Explanation / Answer

Horseradish peroxidase catalyses the oxidation of luminol to 3-aminophthalate via several intermediates. The reaction is accompanied by emission of low-intensity light at 428 nm. However, in the presence of certain chemicals, the light emitted is enhanced up to 1000-fold, making the light easier to detect and increasing the sensitivity of the reaction. The enhancement of light emission is called enhanced chemiluminescence (ECL). Several enhancers can be used such as the commonly known modified phenols (mainly iodo-phenol). However, there are several substrates on the market that use other enhancers which result in luminescence signals up to 13 times greater than phenol-enhanced substrates.[10] The intensity of light is a measure of the number of enzyme molecules reacting and thus of the amount of hybrid. ECL is simple to set up and is sensitive, detecting about 0.5 pg nucleic acid in Southern blots and in northern blots. Detection by chemiluminescent substrates has several advantages over chromogenic substrates. The sensitivity is 10- to 100-fold greater, and quantifying of light emission is possible over a wide dynamic range, whereas that for coloured precipitates is much more limited

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