For degradation of my biological protein sample I want to prepare
Collagenase,alpha-chymotrypsin and Protease XIV. The solution is 0.5-75U/ml. The unit U/ml is my first time unit. So, can you give me technical advice how to prepare. Thank you
The enzymatic unit, 1 U (μmol/min) is defined as the amount of the enzyme that catalyzes the conversion of one micromole of substrate per minute under the specified conditions of the assay method. When you buy enzymes, you usually are informed of the activity of the enzyme preparation in units/mg in the product data sheet, for example https://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/protease-type-xiv.html. It should also inform you how to prepare the enzyme for the assay, which buffers to use, etc. Look at the websites of the suppliers you use to get the details.
Well- the enzymatic unit is a purely functional description of your enzyme preparation in respect to a particular substrate and reaction condition. As you purify an enzyme, the specific activity (U/mg protein) will increase until a maximum is reached for the pure enzyme, therefore the specific activity is used to quantify the effectiveness of the different steps in an enzyme purification scheme. If for a pure enzyme, if you know the specific activity and the molecular weight of the enzyme, you can use this to calculate how many substrate molecules are converted per enzyme molecule per second. See https://innovabiosciences.com/images/stories/innova/pdfs/Enzyme_unit_definitions_and_assay_design.pdf. On the other hand, if you know the specific activity of a pure enzyme (U/mg), you can use the measured activity (U/ml) to calculate the concentration of your enzyme (mg/ml)
If you need to use your proteases to process a biological sample, you want to use just enough of your protease to do the job in a reasonable time frame. From an estimate of the amount of protein in your sample (the micromolar concentration of cleavage sites), you can calculate the amount of protease you need to use for the reaction to complete, for example, in half an hour.
To measure enzymatic activity, you need to be able to determine the concentration of either the substrate or the product as a function of time. This is easiest to do if you can follow the reaction with optical methods (absorbance or fluorescence), therefore assay kits for measuring protease activity use artificial substrates that are not just peptides, but generate a signal that is easy to follow. There are many such systems (see Chapter Protease Assays
), what exactly you need to do to reliably quantitate molar concentrations and reaction speeds from the measured signal depends on the specific assay system you use. Carefully read and follow the instructions for your assay kit!
I have no experience with working in heterogeneous systems (films). However, here are some papers that may help you:
Article Immobilized protein films for assessing surface proteolysis kinetics
Article Kinetics of Adsorption and Proteolytic Cleavage of a Multila...
https://books.google.ch/books?id=oa8YpRsD1kkC&pg=RA3-PA66&lpg=RA3-PA66&dq=proteolytic+cleavage+of+protein+film&source=bl&ots=ld061jbHhj&sig=ACfU3U2P0I4Ba3lVqgekN30ryRy2knjAfg&hl=en&sa=X&ved=2ahUKEwj7tpmq6M7gAhXS_aQKHYlVCEY4ChDoATABegQICBAB
Generally, you dissolve your enzymes in buffered aqueous systems. The data sheet for your specific enzymes will list optimal conditions (pH, maybe metal requirements, e.g. for collagenase). The datasheet for proteas XIV I linked to in my first answer specifies: "The product dissolves in 0.01 M sodium acetate with 0.005 M calcium acetate at pH 7.5 at 37°C; at 0.2 mg/mL the clear solution ranges from colorless to light tan. Calcium ion is recommended for protection from autolysis. The activity of a dilute enzyme solution containing 0.01 to 0.1 M calcium ion was stable over 24 hours at neutral pH at 2-8°C. Protease Type XIV is stable at 4°C for at least six months. Stock solutions of 5 to 20 mg/mL in water are usually stored at 20°C."
Be aware that proteases can also auto-digest in solution and therefore loose activity with time. In the example cited above, Calcium ions inhibit the enzyme. Generally, if you need to store an enzyme stock solution, aliquot and freeze at -20°C.
For very basic protease lab experiments, google "protein digestion lab", you'll find both instruction pdfs and youtube movies.
To assay how much protein has been mobilized from your film (dissolved or digested), you can use a colorimetric assay, such as the biuret assay https://en.wikipedia.org/wiki/Biuret_test , http://biochemistrygirls.blogspot.com/2013/04/experiment-2-protein-experiment.html or bradford assay https://bio-protocol.org/bio101/e45. However, you would also need to find an assay to test at what level your film reaches the desired properties. You would probably use low enzyme concentrations and test after different incubation times.
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