Dynein Dynasty Team's Project

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Background

As catalysts, enzymes serve to facilitate chemical reactions. Two specific enzymes, glucose oxidase and horseradish peroxidase, trigger related reactions in which one's products may act as the other's substrate. Specifically H202, the product of the reaction catalyzed by glucose oxidase, is also the substrate in a colorimetric reaction which is catalyzed by horseradish peroxidase. Although the individual enzymes accelerate the reactions to a point, we believe that the velocity of the reaction and the rate of turnover can be further increased by binding the two enzymes together.

The Purpose (Mission)

Explore the possibilities of making enzymes more efficient by linking them together.

In our project, we test the effect that crosslinking enzymes have on a colorimetric stepwise reaction. Glucose Oxidase (GOX) and Horseradish Peroxidase (HRP) follow the stepwise reaction below:

    H20 + O2 +   Glucose ---(GOx)--> D-Glucose + ABTS + H202 ---(HRP)-->  ABTS (colored) + H20

It is hypothesized that due to the close proximity of the artificially linked enzymes, the rate of reaction for the linked enzymes will be higher than the rate of reaction for the unlinked enzymes.

Methods

Stock Solution Creation

1. 100 mg of Peroxidase from horseradish (lyophilized powder, beige, Sigma Aldrich 77332) was dissolved in 100 mg of deionized water. and separated into 100 total 1 mL aliquots.
2. Each aliquot was kept inside freezer (-20 degrees C). One aliquot was chosen for each experiment. 
3. Stock solutions of Peroxidase and Glucose Oxidase (lyophilized powder, yellow, Sigma Aldrich G2133) were created prior to experiment.
1. Glucose oxidase (GOX) powder was dissolved in 1x TBS, and the concentration was found via a UV-Vis Spectrophotometer (absorption spectrum analyzed at 452 nm with extinction coefficient 28,200).
2. A 1 mL aliquot of Horseradish Peroxidase (HRP) was dissolved in 1x TBS and the protein concentration was verified via a UV-Vis Spectrophotometer (absorbtion spectrum analyzed at 403 nm with extinction coefficient 102,000).

Enzyme Kinetic Coefficient assay

1. Using the stock solution molarity obtained via the UV-Vis Spectrophotometer, a kinetic assay was done for each enzyme, where one enzyme and it's reactants would be saturated while the other had a variable amount of substrate.
1. The molarity of the enzyme used in the assay was calculated to be 10 nM whereas the substrate for the enzyme was generally in the order of milimoles.
2. The colorimetric reaction was run under Kinetic settings in a UV-Vis Spectrophotometer, where the absorption values at the 414 nm peak (colored ABTS absorption peak, extinction coefficient of 36,000) were observed and recorded every 30 seconds. From this data, the reaction rate was obtained relative to the substrate amount.
3. Data was plotted onto a Michaelis Menton Curve and a Lineweaver Burke Plot via the line of Best Fit in Microsoft Excel. 
4. Enzyme kinetic information such as Vmax, Km, and kcat were extrapolated from the Lineweaver Burke plot. 

Enzyme Colorimetric Reaction Model

1. Using the kinetic rate constants obtained via the Enzyme Kinetic Coefficient Assay, a model of our reaction was approximated (an explicit solution could not be found via Mathematica and Matlab without the use of assumptions).
2. The Model was then tested against reaction rates for Enzyme assays with varying amounts of substrate to test the model's accuracy.

NHS Ester Crosslinking

1. 22 mg of Alkyne-PEG5-NHS Ester (100 mg, liquid in glass bottle, Thermo Scientific PI26131) was dissolved in 2997 microliters of Dimethyl Sulfoxide (DMSO) to create a 10 mM solution.
2. 10 mg of Azide-PEG12-NHS Ester (10 mg, liquid in glass bottle, Sigma Aldrich 764191) was dissolved in 2490 microliters of Dimethyl Sulfoxide (DMSO) to create a 10 mM solution.
3. NHS Ester reagent was added to stock solutions of GOX and HRP such that the NHS ester has 20-50 times the molar concentration of the enzymes, which was determined by UV-Vis.
1. Concentrations and volumes were chosen such that the end molarity of both enzymes would be equivalent (320 nM). 
2. 2 mL of each product (GOX-alkyne, GOX-azide, HRP-alkyne, HRP-azide) was created, quenched with 1x TBS, and purified using a Slide-A-lyzer Dialysis Cassette (MW: 2000) .

Huisgen's Azide-Alkyne Cycloaddition Reaction

1. After purification and analysis via a UV-Vis spectrophotometer, the reactants could be combined into ever possible permutation: (Gox-alkyne with GOX-azide, Gox-alkyne with HRP-azide, HRP-alkyne with GOX-Azide & HRP-alkyne with HRP-Azide).
2. The reaction could then be incubated at 98 degrees for a period of 18 hours.
1. Unfortunately, as not only were there few pieces of equipment in the lab that allowed for such a high temperature, such a high temperature would denature all of the proteins and render them useless for future study.
2. The solution was to order some cheap copper catalyst, allowing the reaction to proceed at room temperature with much higher yield.
1. Unfortunately, we are still waiting on the copper catalyst.

Rate Comparison

1. After the Huisgen's cycloaddition reaction is complete, the linked enzymes can be tested in an enzymatic assay against enzymes with equivalent molarity.