Proteolytic+Enzymes



Key words: Proteolytic enzymes, gelatin, collagen, denature, actinidin, kiwifruit, protein
 * PROTEOLYTIC ENZYMES AND KIWI.** **Kjersten.** This experiment aims to show the effects of Proteolytic enzymes on proteins and, subsequently, the effects of extreme temperatures on the proteins themselves. Petri dishes filled with gelatin, a derivative of collagen, were acted upon by kiwifruit samples overnight. Some samples were fresh and used as controls; others had been frozen or boiled for two, four, six, eight or ten minutes. The mass of digested gelatin was taken the next day. The results ultimately reflect the effectiveness of the actinidin, the kiwi’s Proteolytic enzyme, under normal conditions and after exposure to extreme temperatures. The control test came up with a high yield of denatured gelatin, and the frozen kiwi was one in the same. But the boiled samples faired differently: the longer a sample was boiled and, thus, the more time had been allotted for the actinidin to be denatured, the less gelatin melted. The data is far from perfect or even completely consistent, but it comes together to create a clear larger pattern.

Results
Kiwi samples were placed onto petri dishes - numbered one through twenty-five - overnight. The gelatin was near identical but the kiwi used was not. The first three were controls and used fresh, untreated fruit. Others encountered samples that had been boiled and cooled or frozen and thawed, all in an attempt to denature kiwifruit's proteolytic enzyme, actinidin. Freezing the samples had no negative effect on their potency: the frozen trials averaged out at 19.09% percent gel liquefied for each gram of kiwi used, while the controls had an average of 18.76 in the same category. Yet the heated kiwi proved weaker with each passing moment in the boilnig water bath. Two minutes left 21.15 percent liquefied per gram and ten minutes yielded only 5.15%. The results are as follows: heating of kiwifruit effectively denatures actinidin while freezing it does not.

Control:
 * dish # || Mass of gelatin || mass of kiwi (g) || mass liquefied gel || percent liquefied gel || Percent liquefied per gram of kiwi || average percent liquefied per gram of kiwi ||
 * 1 || 4.53g || 1.02g || 0.91g || 20.09 || 19.69% /g || 18.76% /g ||
 * 2 || 4.89g || 1.17g || 1.25g || 25.56 || 21.85% /g ||  ||
 * 3 || 4.46g || 1.05g || 0.69g || 15.47 || 14.73% /g ||  ||

Test 2: boiling (take 1)
 * Dish # || Minutes Boiled || Mass of gelatin || mass of kiwi || mass liquefied gel || percent liquefied percent liquefied per gram of kiwi || average percent liquefied per gram of kiwi ||
 * 8 || 2 min || 5.02  || 1.08 || .58g || 10.70%/g || 13.07 ||
 * 9 || 2 min || 4.24  || .84 || .55g || 15.44%/g ||   ||
 * 6 || 4 min || 5.11  || .91 || 5.11 || 5.76%/g || 14.69 ||
 * 7 || 4min || 4.1  || 1.10 || 4.10 || 26.62 ||   ||
 * 4 || 6 min || 4.79 g  || 1.36 || 5.11 || 8.64% /g || 9.71 ||
 * 5 || 6 min || 4.37 g  || .63 || 4.1 || 9.78% /g ||   ||

Test 3: freezing
 * Dish number || mass gel || mass kiwi || mass liquefied gel || percent liquefied || percent liquefied per gram of kiwi || average percent liquefied per gram of kiwi ||
 * 10 || 4.43 || 1.23 || .90 || 20.32 || 16.52 || 19.09 ||
 * 11 || 4.32 || 1.10 || 1.04 || 24.07 || 21.89 ||  ||
 * 12 || 3.94 || 1.17 || .87 || 22.08 || 18.87 ||  ||

Test 4: Boiling (take 2)

 * Dish # || Time boiled || Mass gel || Mass Kiwi || Mass liquefied Gel || Percent liquefied || Percent liquefied per gram of kiwi || Average percent liquefied per gram ||
 * 13 || 2 min || 3.85 || 1.01 || 0.45 || 11.69 || 11.57 || 21.15 ||
 * 14 || 2 || 3.68 || 1.3 || 1.47 || 39.95 || 30.73 ||  ||
 * 15 || 4 || 3.98 || 1.05 || 0.28 || 7.04 || 6.70 || 7.52 ||
 * 16 || 4 || 3.85 || 0.84 || 0.27 || 7.01 || 8.35 ||  ||
 * 17 || 6 || 4.34 || 1.24 || 0.21 || 4.84 || 3.90 || 4.48 ||
 * 18 || 6 || 4.11 || 1.3 || 0.27 || 6.57 || 5.05 ||  ||
 * 19 || 8 || 3.85 || 0.97 || 0.2 || 5.19 || 5.36 || 6.97 ||
 * 20 || 8 || 4.15 || 0.93 || 0.24 || 5.78 || 6.22 ||  ||
 * 21 || 8 || 3.57 || 0.93 || 0.31 || 8.68 || 9.34 ||  ||
 * 22 || 10 || 4.27 || 1.1 || 0.22 || 5.15 || 4.68 || 5.15 ||
 * 23 || 10 || 4.25 || 1.07 || 0.18 || 4.24 || 3.96 ||  ||
 * 24 || 10 || 4.02 || 0.95 || 0.26 || 6.47 || 6.81 ||  ||

Article Summary
The article outlines an experiment that shows that collagenase, a proteolytic enzyme, unwinds the triple-helical structure of collagen before hydrolysis occurs at specific peptide bonds. The authors first observed the vulnerability of native vollagen to cleavage by MMP-1 ( gene that encodes interstital collagenase) at different temeratures. They found that the gelatin's bonds were cleaved much more easily at lower temperatures, for conditions below 25 degres C made the helix's backbone more suceptible to unwinding. According to their findings, undoing a triple helix my actually be a necessary first step in the breakdown of collagen by collagenase - this structure is expecially strong and makes it harder for a protease to pick it apart. They also determined that MP-1 only unwinds collagen I in one segment of the helix - it does not influence the general triple-helical structure. Through their studies they claim that after unwinding, the collagenase takes apart each of the three strands one at a time. A divide and conquer strategy of sorts, which may very well apply to the breakdown of gelatin, a derivative of collagen, and proteases. Chung, Linda, Deendayal Dinakarpandian, Naoto Yoshida, Janelle Lauer-Fields, and Gregg Fields. “Collagenase unwinds triple-helical structure prior to peptide bond hydrolysis.” //Embo Journal// 23.15 (2004): n. pg. Web. 8 Feb 2010. <__http://www.ncbi.nlm.nih.gov/pmc/articles/PMC514933/?tool=pmcentrez__>.