PROTEASE

The use of meat tenderizer enzyme that only "clips" certain peptide bonds.

| SUPPLIES |

Proteins are snipped (hydrolyzed) by enzymes collectively called proteases. There are a great number of different proteases - each able to cut proteins in its own special way. Some start at the end of a protein chain and cut off one amino acid at a time. These are called exopeptidases, because "exo-" means 'outside, and these cut in from the outside. Other proteases are called endopeptidases because they snip the peptide bonds found deep within a polypeptide. Usually endopeptidases are highly specific in what they do. Meat tenderizer contains a plant protease called papaine, which only hydrolyzes the peptide bonds next to aromatic amino acids like tyrosine and phenylalanine (the carboxyl group of those aromatic amino acids is freed). Chymotrypsin is the normal animal digestive enzyme which acts the same way as do plants' papaine.

Other proteases: trypsin is a stomach endo-protease that frees the carboxyl groups of basic amino acids such as lysine and arginine. Still other proteases are extremely specific and only make one snip in only one kind of protein. Two examples of these are the proteases that snip off pieces from pro-insulin and from fibrinogen to "activate" two powerful proteins that are needed in large amounts almost instantly. Respectively, insulin (to control blood sugar levels right after you have consumed some candy) and fibrin (to cause your blood to clot at the site of a wound). (Soon you will be learning about gene expression resulting in the making of proteins. You will be learning a little about post-translational modifications of proteins. Fibrin and insulin are two such examples of this.)

Today we are going to run a delicious experiment using the papaine found in meat tenderizer. Gelatin desserts congeal into a gel because the gelatin (a derivative of collogen - the protein that is used to make tendons, and also makes your ears and nose somewhat stiff) tries to get back with itself like it was in collogen. However, it doesn't quite figure out how to do it and makes a gel instead. Obviously, if something were to snip the gelatin strands, it would not be able to gel. So what you are going to do is dissolve various amounts of papaine in warm "Jello" and then see which prevent the gelling from occurring.

1. Prepare your papaine: measure out a level 1/8th teaspoon of meat tenderizer.
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2. Set up a series of six test tubes as shown. The first one has 4 ml of water in it and the others have 2 ml in them. Dump the meat tenderizer into the first tube; thoroughly mix to dissolve. Then begin a "serial dilution:" transfer 2 ml from the first tube to the second, mix. Then transfer 2 ml from the second tube to the third, and so on. Discard 2 ml from the fifth tube. (The sixth tube is your "control" as it will have no papaine in it.

   
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3. Put each little batch into a separate and labelled test tube. Add another tube as a control (no papaine in it).
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4. Make sure to use a delicious flavor of Jello. You will have a snack after the experiment as there will be unused leftovers.
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5. Using a pan you have brought from home (not a mercury-contaminated lab pan), make up the Jello by only partially following the directions on the package (1 cup = 225 ml).
   1. Bring one cup of water to a boil
   2. Stir in one small package of Jello, and keep stirring for 2 minutes to make sure all the gelatin granules are dissolved.
   3. Pour molten Jello from the pan into a 3-cup (or larger) measuring cup. (After this point you will deviate from the directions on the box.)
   4. Add crushed ice with stirring up to the 2-cup line. (A 3-cup measuring cup was used to give you room to splash and stir.)
   5. Keep stirring until all ice chunks have melted.
   6. IMMEDIATELY go to the next step!
   
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6. Get the rack of tubes with papaine dilutions you made earlier. Fill each tube 1/3 to the top with the cooling Jello you have made, and then IMMEDIATELY swirl the tubes to thoroughly dissolve and distribute the enzyme.
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7. Allow the tubes to incubate at room temperature for 15 to 20 minutes, then
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8. Place your rack of tubes in ice-water.
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9. Once your control tube has gelled (it can be inverted without its draining out of the tube), see what has happened to the gelling in the other tubes, and record your results.
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10. Meanwhile place the extra Jello in the refrigerator for gelling and later snack.
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11. When the pan of extra Jello has congealed, get out the paper bowls and plastic spoons, give everyone a share of it, and take it to the designated non-lab area to enjoy.
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