Investigating the Effects of Proteolytic Enzymes

Investigating the Effects of Proteolytic Enzymes on Collagen Based Gelatin Background Information Your Most Plentiful Protein About one quarter of all of the protein in your body is collagen. Collagen is a major structural protein, forming molecular cables that strengthen the tendons and vast, resilient sheets that support the skin and internal organs. Bones and teeth are made by adding mineral crystals to collagen. Collagen provides structure to our bodies, protecting and supporting the softer tissues and connecting them with the skeleton. But, in spite of its critical function in the body, collagen is a relatively simple protein. The Collagen Triple Helix Collagen is composed of three chains, wound together in a tight triple helix. Theillustration included here shows only a small segment of the entire molecule -- each chainis over 1400 amino acids long and only about 20 are shown here. A repeated sequence of three amino acids forms this sturdy structure. Every third amino acid is glycine, a small amino acid that fits perfectly inside the helix. Many of the remaining positions in the chain are filled by two unexpected amino acids: proline and a modified version of proline, hydroxyproline. We wouldn’t expect proline to be this common, because it forms a kink in the polypeptide chain that is difficult to accommodate in typical globular proteins. But, as you can see on the next page, it seems to be just the right shape for this structural protein. Vitamin C Hydroxyproline, which is critical for collagen stability, is created by modifying normal proline amino acids after the collagen chain is built. The reaction requires vitamin C to assist in the addition of oxygen. Unfortunately, we cannot make vitamin C within our bodies, and if we don’t get enough in our diet, the results can be disastrous. Vitamin C deficiency slows the production of hydroxyproline and stops the construction of new collagen, ultimately causing scurvy. The symptoms of scurvy – loss of teeth and easy bruising – are caused by the lack of collagen to repair the wear-and-tear caused by everyday activities. Collagen on the Grocery Shelf Collagen from livestock animals is a familiar ingredient for cooking. Like most proteins, when collagen is heated, it loses all of its structure. The triple helix unwinds and the chains separate. Then, when this denatured mass of tangled chains cools down, it soaks up all of the surrounding water like a sponge, forming gelatin. Bromelain Pineapple is cultivated for fruit, used fresh, canned, frozen, or made into juices, syrups, or candied. Pineapple bran, the residue after juicing, is high in vitamin A, and is used in livestock feed. From the juice may be extracted citric acid, or on fermentation, alcohol. Commercial bromelain is generally prepared from pineapple wastes. Fresh pineapple is rich in bromelain, a group of sulfur-containing proteolytic (protein-digesting) enzymes. A mixture of several proteases, bromelain is used in meat tenderizers, in chill-proofing beer, manufacturing precooked cereals, in certain cosmetics, and in preparations to treat edema and inflammation. Bromelain is also nematicidal (kills plant and animal parasites such as nematodes). Investigating the Effects of Proteolytic Enzymes on Collagen Based Gelatin

Introduction Foods are not only important as nutrients for cells; they are also the cause of some stains on fabrics. Since some of the large complex molecules in food have hydrophobic properties and do not dissolve well in water they are often left on clothes after washing. Enzymes are often added to detergents to break down the large complex molecules removing the stains. In this exercise you will investigate the addition of protease to detergents by their ability to break down the protein found in gelatin, a major part of Knox™ and JELL-O ™. Proteases are naturally found in some fruits such as pineapple, you will also investigate

Purpose You will observe the effect of enzymes in detergents, cleaning solutions, pineapple and meat tenderizer on collagen based Knox™ Gelatin or Jell-O™.

Materials 2 petri plate containing Knox™ Gelatin or JELL-O™ set to a pH of 8 (not edible!) 1 plastic straw section 1 toothpick 1 Sharpie 1 clear metric ruler sample detergents sample cleaning solutions fresh pineapple juice canned pineapple juice fresh fruit juice distilled water a plastic pipette for each sample Procedure 1. Obtain a gelatin filled plate. Label the plate on the bottom by writing (near the edge and in small letters) your name, class period, and the date. 2. Using a piece of plastic soda straw, cut wells in the gelatin as shown in the diagram. 3. Remove the plugs of gelatin from the straw with a toothpick. Take care not to tear the layer of gelatin in the petri dish. Number the wells on the bottom of your plate, so that when face up, the numbers are as shown on the template. (This means you write the numbers backward and counter-- clockwise on the bottom of your plate.) 4. Measure the diameter of the wells (in millimeters) record these measurements in an observation table as "initial diameter" 5. Record the sample name and well number in your observation table. 6. Use only the dropper that is in each solution. Do not exchange droppers between samples. 7. Load the wells by placing the pipette into the well and gently dispense just enough liquid to fill the well. Try not to drop any liquid onto the surface of the gelatin. If you do, note the location of the drop by drawing a picture in your lab book. 8. Load distilled water into well #7 on each plate you use. This will be your control. 9. Carefully load each of the wells with one of the detergent solutions, the fruit juice solutions and the meat tenderizer solution. 10. Replace the lid on the plate. Do not turn the plate upside down. You will spill the solutions that you just loaded into the wells. 11. Let the plate sit undisturbed for several hours or overnight at room temperature. 12. Measure the largest diameter of each well in millimeters. The diameter of a well is the distance from solid gelatin on one side to the solid gelatin on the other side. Record these number in your observation table the "final diameter". 13. Calculate the change in diameter for each sample Your Write Up

Conclusion States a conclusion, with justification, based on a reasonable interpretation of the data (observations) that compares the:  effectiveness of each detergent or cleaning agent by ranking them based on their ability to catabolize the protein collagen.  enzymatic activity of the two different pineapple juice samples and the other fruit juice.  effectiveness of the detergent that showed the greatest amount of enzymatic activity to the meat tenderizer and pineapple juice.

Evaluation Write a statement that evaluates the weaknesses and limitation of this investigation.

Improving the investigation Suggests realistic improvements to this investigation in respect of identified weaknesses and limitations in your evaluation. Appendix 1. Data collection example to be copied into your lab book.

Initial Final Change in Well Sample Diameter Diameter Diameter Number (mm) (mm) (mm)

Room temperature:

Atmospheric Pressure:

Notes: