Team:UCAS-China/Measurement

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• measurement
• BCA
• CPM assay
• HPLC
• Coenzyme
   Stability
• Reaction Kinetics

Bicinchoninic acid (BCA)

We use bicinchoninic acid(BCA) to measure the concentration of protein, therefore we can further verified that we obtained the target protein and analyzed its physicochemical properties.

BCA is a protein assay based on BCA which allows the concentration of protein in solution to be measured. In an alkaline environment, the protein is able to reduce Cu+2 to Cu+. The Cu+ cation is able to chelate two BCA molecules, resulting in a purple colour that can then be measured at 562 nm. The more protein present in the solution, the more intense the colour will be.

CPM assay

We use CPM assay to measure the thermal stability of our enzymes--including wild-type and mutant ones. CPM is a kind of blue fluorescent mercapturic reactive dye known as N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleoylethylamine, which is non-fluorescent in itself but reacts with mercapturic groups to produce fluorescence.

At lower temperatures, the cysteine residues in the protein tend to form disulphide bonds, which are stable within the hydrophobic interior of the protein. At this point no sulfhydryl groups are exposed and CPM does not show fluorescence. When the temperature rises to a certain level, the tertiary structure of the protein is disrupted, the cysteine residues are exposed and the CPM reacts with the sulfhydryl groups to show fluorescence.

Using this principle, we can tell the thermal stability of a protein by simply measuring the change in fluorescence intensity while changing the temperature.

HPLC

We use the principles of high performance liquid chromatography to analyse organic compounds with high boiling points that are not volatile, heat unstable and have a high molecular weight.

The mobile phase in the reservoir is pumped into the system by a high-pressure pump. The sample solution enters the mobile phase through the injector and is loaded onto the column (stationary phase) by the mobile phase, and as the components of the sample solution have different partition coefficients in the two phases, they are separated into individual components and flow out of the column in turn after repeated adsorption-desorption partitioning processes.

When using HPLC to study the concentration of substances under changing conditions, We need to first perform an HPLC analysis of a known concentration of the substance in order to obtain a correspondence between the concentration and the chromatographic peak area, and then plot a standard curve.

Coenzyme Stability

HPLC can be used for SAM and CoQ0 concentration measurements. With the help of HPLC analysis, we measured the concentration of coenzymes stored at the same time and at different temperatures to get an idea of their thermal stability.

Reaction Kinetics

HPLC can also be used for caffeine and theacrine concentration measurements. We verified the effectiveness of the cell-free caffeine degradation system by measuring the concentration of caffeine and theacrine in solution, and further measured the reaction rate of the enzymatic reaction.