Effects of Heavy Metal Ions on the Action of Trypsin Enzyme - Coursework Example

? Research has shown that, the Metallic Ions like the Mercury ions and Lead Ions do take part in some reactions resulting into the inhibition of trypsin; in order for this test to take place it was very relevant to do an inclusion of the test such as Mann Whitney U-test to ascertain the above claims. It has also been realized that the Histidine acts as an active center of the enzyme trypsin which do bind with the heavy metal ions, which consequently results into the inhibition of enzymatic action. In this particular study, Mercury ions and Lead ions was added to milk containing the Trypsin, the result shown more clearly, that the Metallic ions in high concentrations do have a negative impact on the actions of the enzyme. The above observation will get into the discussion in association with the dissociation of Mercury Nitrate and the lead Nitrate. The above observations might also be explained in terms some complex reactions which do involving the enzyme trypsin and metal ions. RESEARCH AND RATIONAL Enzymes are biological catalyst made up of proteins, they speed up the rate of chemical reactions by lowering the activation energy hence providing an alternative pathway (fig.1). Enzymes remain unchanged at the end of a reaction. They are classified as globular proteins, they are made up of polypeptide chains which coil and or fold up to give a 3D structure which determines the shape of the enzyme and hence, the shape of the active site. http://tfscientist.hubpages.com/hub/what-are-enzymes-where-do-they-work Figure 1http://www.biologyguide.net/unit1/2_enzymes.htm All enzymes have an active site, in 1814 Emil Fischer proposed the lock and key model. According to this theory, the substrate fits perfectly into the enzymes active site hence forming an enzyme substrate complex, causing the bonds in the substrate to change. This will eventually lead to the formation of products. The products are released from the enzyme active site leaving the enzyme free to accept another substrate. http://www.elmhurst.edu/~chm/vchembook/571lockkey.html The diagram below illustrates this theory. Figure 2: http://en.wikipedia.org/wiki/File:Competitive_inhibition.svg However X-ray crystallography and computer assisted modelling, research has shown that the lock and key model is not accurate. This has led to the introduction of the ‘induced-fit theory’. It assumes that the substrate influences the final shape of the enzyme active site and that the active site is malleable. Only specific substrates will be able to alter the active site slightly in order for a reaction to take place [1].The diagram below illustrates the induced fit theory. Figure 3: http://www.biologyguide.net/unit1/2_enzymes.htm There are various factors that influence the activity of enzymes, these include; pH, temperature and Inhibitors. Inhibitors are substances that affect the activity of enzyme, if the site which active of the enzyme gets occupied by a substance which is not a substrate, the activity of the enzyme will decrease because the substrate cannot bind to the active site. This means that both the substrate and the molecule are competing for space on the active site. This is known as a competitive inhibition and can be reversed by the addition of more substrate.  Non-competitive inhibition is another form of inhibition where a molecule binds to the allosteric site on an enzyme hence changing the shape of the active site. This prevents the substrate from binding to the active point. Usually this type is reversible but cannot be overcome by increasing substrate concentration. Trypsin is a serine protease found in the human digestive system, it is essential for the hydrolysis of protean such as casein found in milk http://www.princeton.edu/~achaney/tmve/wiki100k/docs/Trypsin.html. Without trypsin, it would be difficult for the human body to absorbed protein; Pb (NO3 )2 and Hg (NO3 )2 contain Pb2+ and Hg2+ ions respectively. These meal ions acts as non-competitive inhibitors and this means that there will be fewer successful collisions between the enzyme and casein in milk hence fewer enzyme substrate complex will be formed. Thus increasing the time taken for the hydrolysis of casein. The extent of inhibition will therefore depend on the concentration of the heavy metal ions. In this experiment, the effect of two different concentrations (0.1 mol dm-3 and 0.0l mol dm-3) lead nitrate and mercury nitrate will be compared by recording the readings on the colorimeter for four minutes. The milky colour should gradually fade hence the readings on the colorimeter will decrease. Therefore my experimental hypothesis is that an increase in concentration of the metallic ions will decrease the activity of trypsin on casein in milk. This is a directional hypothesis, therefore the test is one tailed. PLANNING Directional Hypothesis: An increase in the concentration of the heavy metal ions (Lead nitrate and Mercury nitrate) will decrease the activity of Trypsin on Casein. Null Hypothesis: Increasing the concentration of Lead nitrate and Mercury nitrate will have no effect on the activity of Trypsin. Independent Variable: The different concentrations of Lead nitrate and Mercury nitrate. Dependant Variable: The change in the value of absorbance on the colorimeter after the heavy metal ion solute has been added to the milk; from 15 seconds to 240 seconds. Pilot study: Chemicals Required: a) 1% Porcine Trypsin b) 2% Reconstituted milk (contains the protein casein) c)  0.1 mol dm-3 and 0.0l mol dm-3 Lead nitrate solution d) 0.1 mol dm-3 and 0.0l mol dm-3 Mercury nitrate solution e) Distilled water Apparatus: a) Colorimeter b) Test tubes c) Test tube rack d) Stop watch e) 100 cm3 Volumetric flasks f) 10 cm3 pipettes and 5cm3 pipettes g) Pipette fillers  h) Thermometer Method Make up 1% Trypsin by dissolving 0.50grams of the solid in 50cm3 of distilled water. Make up 2% reconstituted milk by dissolving 2.00grams of milk in 100cm3 of distilled water. Measure out 3cm3 of milk into a test tube, followed by 1cm3 of Trypsin and 2 cm3 of the inhibitor. Start the stop watch as soon as the enzyme solution comes in contact with the milk. Mix the solution with a glass rod. The first reading is taken at 15 seconds, the second reading is taken at 30 seconds, and all other successive readings are taken at regular intervals of 30 seconds, till the time reaches four minutes. My first run was done without any inhibitor, it took 30 seconds for reading on the colorimeter to drop to 0.00 Arbitrary unit, which was too fast. As a result of this, I decided to increase the milk concentration to 5%, this time, it took 60 seconds which was still too fast. As a result of this, I decided to reduce my Trypsin concentration to 0.5%. Results Gotten From My Pilot Study  Absorbance (arbitrary units) at; Inhibitor concentration Pb (NO3 )2 15s 30s 60s 90s 120s 150s 180s 210s 240 s 0.00 1.65 1.56 1.23 0.14 0.00 0.00 1.67 1.56 1.29 0.49 0.00 0.00 1.64 1.54 1.21 0.22 0.00 0.00 1.69 1.60 1.31 0.66 0.00 0.00 1.66 1.55 1.23 0.32 0.00 0.00 1.62 1.53 1.21 0.41 0.00 Absorbance (arbitrary units) at; Inhibitor concentration Pb (NO3 )2 15s 30s 60s 90s 120s 150s 180s 210s 240 s 0.01 1.65 1.61 1.52 1.41 1.26 1.07 0.80 0.44 0.07 0.0l 1.62 1.58 1.48 1.36 1.19 0.95 0.63 0.24 0.00 0.01 1.63 1.59 1.50 1.38 1.23 1.02 0.74 0.39 0.01 0.01 1.60 1.56 1.45 1.32 1.16 0.91 0.62 0.26 0.00 0.0l 1.64 1.60 1.52 1.41 1.27 1.08 0.81 0.47 0.08 0.01 1.64 1.61 1.53 1.44 1.33 1.180 0.98 0.72 0.41 Absorbance (arbitrary units) at; Inhibitor concentration Pb (NO3 )2 15s 30s 60s 90s 120s 150s 180s 210s 240s 0.1 1.76 1.76 1.74 1.74 1.73 1.72 1.71 1.71 1.70 0.1 1.77 1.77 1.76 1.74 1.74 1.73 1.73 1.72 1.71 0.1 1.72 1.72 1.70 1.69 1.68 1.68 1.67 1.66 1.66 0.1 1.77 1.76 1.75 1.73 1.72 1.71 1.70 1.69 1.69 0.1 1.80 1.80 1.78 1.77 1.76 1.75 1.75 1.75 1.73 0.1 1.81 1.80 1.79 1.78 1.77 1.76 1.75 1.75 1.74 The results from my pilot study support my directional hypothesis. However, I have decided to increase my range of concentration and reduce the volume of trypsin and milk added by 0.5 cm3. I have also decided to do a blank run which will contain no inhibitor solution and no trypsin (distilled water is used instead). (why) . I will also continue to do six repeats for each concentration, this will ensure that my results are accurate and hence reliable. For my statistical test, I will be using the Mann-Whitney U test. The Mann Whitney U test is applied looking for a difference between two sets of independent data where the distribution is not normally distributed Revised Method  Chemicals Required:  a) 0.5% Porcine Trypsin b) 5% Reconstituted milk (contains the protein casein) c)  0.1 mol dm-3, 0.0l mol dm-3, 0.001 mol dm-3 and 0.0001 mol dm-3 Lead nitrate solution d) 0.1 mol dm-3, 0.0l mol dm-3, 0.001 mol dm-3 and 0.0001 mol dm-3Mercury nitrate solution e) Distilled water Method Measure out 2.5 cm3 of 5% reconstituted milk into a test tube and label it A. Measure out 2 cm3 of the inhibitor solution and 0.5 cm3 of trypsin into another test tube and label it B. Pour the mixture in the test tube B into the test tube A, then start stop watch. Pour the mixture back into the test tube B and place it in a colorimeter. Take readings of light absorbance at 15s and 30s. All other readings are taken at successive intervals of 30s for four minutes (240s). Repeat the above method six times for each concentrations, making sure to zero the colorimeter using a test tube containing distilled water before taking readings for each test tube. Table 1 Results from the Blank run i.e. no trypsin and no inhibitor (Temperature 200c)  Absorbance (arbitrary units) at; 15s 30s 60s 90s 120s 150s 180s 210s 240s Run 1 1.80 1.80 1.80 1.80 1.80 1.80 1.79 1.79 1.79 Run 2 1.68 1.68 1.68 1.68 1.68 1.68 1.68 1.68 1.69 Run 3 1.72 1.72 1.72 1.72 1.72 1.72 1.72 1.72 1.72 Run 4 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.69 Run 5 1.72 1.72 1.71 1.71 1.71 1.71 1.71 1.71 1.71 Run 6 1.72 1.72 1.72 1.72 1.72 1.72 1.72 1.72 1.72 Average 1.72 1.72 1.72 1.72 1.72 1.72 1.72 1.72 1.72 Table 2 Results without any inhibitor (Temperature 200c)  Absorbance (arbitrary units) at; No inhibitor 15s 30s 60s 90s 120s 150s 180s 210s 240s Run 1 1.70 1.66 1.57 1.45 1.30 1.06 0.68 0.15 0.00 Run 2 1.69 1.65 1.55 1.42 1.26 0.98 0.56 0.00 Run 3 1.69 1.65 1.56 1.44 1.29 1.03 0.65 0.13 0.00 Run 4 1.67 1.62 1.52 1.37 1.16 0.82 0.30 0.00 Run 4 1.64 1.61 1.52 1.41 1.27 1.06 0.74 0.30 0.00 Run 6 1.70 1.66 1.57 1.45 1.30 1.07 0.71 0.19 0.00 Average 1.68 1.64 1.55 1.42 1.26 1.00 0.61 0.13 0.00 Lead Nitrate results Table 1 shows the results for 0.0001mol dm-3 Lead nitrate (Temperature; initial 220c -final 250c)  Absorbance (arbitrary unit) at; 0.0001mol dm-3 15s 30s 60s 90s 120s 150s 180s 210s 240s Run 1 1.58 1.53 1.41 1.21 0.85 0.22 0.00 Run 2 1.62 1.58 1.48 1.33 1.08 0.63 0.00 Run 3 1.69 1.55 1.43 1.27 0.98 0.47 0.00 Run 4 1.55 1.49 1.31 0.98 0.36 0.00 Run 5 1.56 1.50 1.34 1.06 0.50 0.00 Run 6 1.51 1.43 1.19 0.68 0.00 Average 1.57 1.51 1.36 1.09 0.22 0.00 0.00 Run 4, 5 and 6 are anomalies as they are faster than expected. The likely reason is for the increase in temperature by 30c i.e. from 22 0c to 250c. An increase in the temperature resulting into in an increase in the kinetic energy and hence more successful collision between the substrate (casein) and the enzyme (trypsin), this means the time taken for the reaction is reduced. http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm Table 2 shows the results for 0.001mol dm-3 Lead nitrate. (Temperature 250c)  Absorbance (arbitrary units) at;  0.001mol dm-3 15s 30s 60s 90s 120s 150s 180s 210s 240s Run 1 1.73 1.69 1.60 1.49 1.33 1.06 0.59 0.01 0.01 Run 2 1.71 1.67 1.58 1.44 1.23 0.82 0.18 0.00 Run 3 1.71 1.67 1.57 1.44 1.24 0.84 0.23 0.00 Run 4 1.77 1.73 1.65 1.54 1.39 1.14 0.69 0.08 0.00 Run 5 1.70 1.66 1.55 1.40 1.14 0.64 0.00 Run 6 1.74 1.70 1.61 1.49 1.32 1.00 0.46 0.00 Average 1.73 1.69 1.59 1.47 1.23 0.92 0.36 0.02 0.00 The anomaly in Run 1 at 240s is likely to be caused by an error in the colorimeter. By 240 s the absorbance value should have dropped to 0.00 arbitrary unit. Table 3 shows the results for 0.0lmol dm-3 Lead nitrate. (Temperature 250c)  Absorbance (arbitrary units) at; 0.01mol dm-3 15s 30s 60s 90s 120s 150s 180s 210s 240s Run 1 1.84 1.83 1.83 1.82 1.81 1.80 1.80 1.79 1.79 Run 2 1.85 1.84 1.83 1.83 1.82 1.81 1.81 1.80 1.80 Run 3 1.83 1.83 1.82 1.81 1.80 1.80 1.79 1.79 1.78 Run 4 1.88 1.88 1.87 1.86 1.85 1.85 1.84 1.84 1.83 Run 5 1.80 1.79 1.78 1.78 1.77 1.76 1.76 1.75 1.75 Run 6 1.83 1.83 1.82 1.81 1.80 1,80 1.79 1.78 1.79 Average 1.84 1.83 1.83 1.82 1.81 1.80 1.80 1.79 1.79 Table 4 shows the results for 0.1mol dm-3 Lead nitrate (Temperature; initial 250c – final 230c)  Absorbance (arbitrary unit) at; 0.1mol dm-3 15s 30s 60s 90s 120s 150s 180s 210s 240s Run 1 1.84 1.84 1.83 1.82 1.82 1.82 1.82 1.82 1.82 Run 2 1.78 1.78 1.78 1.78 1.78 1.78 1.78 1.78 1.78 Run 3 1.82 1.82 1.81 1.81 1.81 1.81 1.81 1.81 1.81 Run 4 1.82 1.81 1.81 1.80 1.80 1.80 1.80 1.80 1.80 Run 5 1.79 1.79 1.78 1.78 1.78 1.78 1.78 1.78 1.78 Run 6 1.73 1.73 1.72 1.72 1.73 1.73 1.73 1.73 1.73 Average 1.80 1.80 1.80 1.80 1.80 1.80 1.80 1.80 1.80 Table 5 shows the average results for each concentration of Lead nitrate  Absorbance (arbitrary units) at; Concentration of Pb (NO3 )2 15s 30s 60s 90s 120s 150s 180s 210s 240s No inhibitor 1.68 1.64 1.55 1.42 1.26 1.00 0.61 0.13 0.00 0.0001mol dm-3 1.57 1.51 1.36 1.09 0.22 0.00 0.001mol dm-3 1.73 1.69 1.59 1.47 1.23 0.92 0.36 0.02 0.00 0.01mol dm-3 1.84 1.83 1.83 1.82 1.81 1.80 1.80 1.79 1.79 0.1mol dm-3 1.80 1.80 1.80 1.80 1.80 1.80 1.80 1.80 1.80 Mercury Nitrate Results Table 1 shows results for 0.0001mol dm-3 Mercury nitrate (Temperature; initial 230c – final 220c)  Absorbance (arbitrary units) at; 0.0001mol dm-3 15s 30s 60s 90s 120s 150s 180s 210s 240s Run 1 1.67 1.63 1.52 1.38 1.17 0.80 0.22 0.00 Run 2 1.64 1.60 1.48 1.31 1.06 0.65 0.10 0.00 Run 3 1.68 1.64 1.54 1.40 1.19 0.83 0.29 0.00 Run 4 1.70 1.67 1.59 1.48 1.34 1.10 0.78 0.25 0.00 Run 5 1.67 1.64 1.56 1.45 1.31 1.10 0.77 0.35 0.00 Run 6 1.69 1.66 1.57 1.45 1.28 1.02 0.60 0.04 0.00 Average 1.68 1.64 1.54 1.41 1.23 0.92 0.46 0.12 0.00 Table 2 shows results for 0.001mol dm-3 Mercury nitrate (Temperature 220c)  Absorbance (arbitrary units) at; 0.001mol dm-3 15s 30s 60s 90s 120s 150s 180s 210s 240s Run 1 1.73 1.73 1.70 1.68 1.64 1.61 1.57 1.52 1.47 Run 2 1.70 1.70 1.69 1.67 1.65 1.63 1.60 1.57 1.54 Run 3 1.77 1.77 1.76 1.75 1.73 1.71 1.70 1.68 1.66 Run 4 1.75 1.75 1.74 1.73 1.72 1.71 1.69 1.68 1.66 Run 5 1.76 1.76 1.76 1.75 1.73 1.72 1.71 1.69 1.68 Run 6 1.70 1.70 1.70 1.69 1.68 1.67 1.66 1.64 1.63 Average 1.74 1.74 1.73 1.71 1.69 1.68 1.66 1.63 1.61 Table 3 shows results for 0.01mol dm-3 Mercury nitrate (Temperature 210c)  Absorbance (arbitrary units) at; 0.01mol dm-3 15s 30s 60s 90s 120s 150s 180s 210s 240s Run 1 1.88 1.88 1.88 1.88 1.88 1.88 1.88 1.88 1.88 Run 2 1.89 1.90 1.90 1.90 1.90 1.90 1.90 1.89 1.89 Run 3 1.89 1.89 1.89 1.89 1.89 1.89 1.89 1.89 1.89 Run 4 1.89 1.89 1.89 1.89 1.89 1.89 1.89 1.89 1.89 Run 5 1.85 1.85 1.86 1.86 1.86 1.86 1.86 1.86 1.86 Run 6 1.91 1.91 1.91 1.91 1.91 1.91 1.91 1.91 1.91 Average 1.88 1.89 1.89 1.89 1.89 1.89 1.89 1.89 1.89 Table 4 shows the results for 0.1mol dm-3 Mercury nitrate (Temperature 200c)  Absorbance (arbitrary units) at; 0.1mol dm-3 15s 30s 60s 90s 120s 150s 180s 210s 240s Run 1 1.98 1.98 1.99 1.99 1.99 1.99 1.99 1.99 1.99 Run 2 1.93 1.93 1.94 1.94 1.95 1.95 1.96 1.96 1.96 Run 3 1.96 1.96 1.97 1.98 1.98 1.98 1.99 1.99 1.99 Run 4 1.98 1.99 1.99 1.99 1.99 1.99 1.99 1.99 1.99 Run 5 1.99 1.99 1.99 1.99 1.99 1.99 1.99 1.99 1.99 Run 6 1.98 1.99 1.99 1.99 1.99 1.99 1.99 1.99 1.99 Average 1.97 1.97 1.98 1.98 1.98 1.98 1.99 1.99 1.99 Table 5 shows the average results for each concentration of Mercury nitrate  Absorbance (arbitrary units) at;  Concentration of Hg (NO3 )2 15s 30s 60s 90s 120s 150s 180s 210s 240s No inhibitor 1.68 1.64 1.55 1.42 1.26 1.00 0.61 0.13 0.00 0.0001mol dm-3 1.68 1.64 1.54 1.41 1.23 0.92 0.46 0.12 0.00 0.001mol dm-3 1.74 1.74 1.73 1.71 1.69 1.68 1.66 1.63 1.61 0.01mol dm-3 1.88 1.89 1.89 1.89 1.89 1.89 1.89 1.89 1.89 0.1mol dm-3 1.97 1.97 1.98 1.98 1.98 1.98 1.99 1.99 1.99 GRAPH 1 shows an increase in absorption value as the concentration of lead nitrate increases. GRAPH 2 The graph above (graph 2) shows the lines of best fit for each concentration of Lead nitrate and this allows me to calculate the rate of reaction. This is done by taking the gradient of the line using the formula . Rate of reaction when the lead Nitrate is 0.1mol/dm3 Let’s calculate the rates of reaction when the amount of inhibitor is 0.1mol/dm3 Taking the readings at two different points a (200, 1.57), b(50, 1.57) The gradient/ rate of reaction at that point is =-0.00007 You will realise that the rate of reaction when the concentration of Lead nitrate is 0.1mol/dm3 is 0.0007 per second. Rate of reaction when the lead Nitrate is 0.01mol/dm3 Let’s calculate the rates of reaction when the amount of inhibitor is 0.1mol/dm3 Taking the readings at two different points a (200, 1.57), b(50, 1.57) The gradient/ rate of reaction at that point is =-0.0001 You will realise that the rate of reaction when the concentration of Lead nitrate is 0.1mol/dm3 is 0.0001 per second. Rate of reaction when the lead Nitrate is 0.001mol/dm3 Let’s calculate the rates of reaction when the amount of inhibitor is 0.1mol/dm3 Taking the readings at two different points a (150, 0.59), b(40, 1.50) The gradient/ rate of reaction at that point is =-0.0091 You will realise that the rate of reaction when the concentration of Lead nitrate is 0.1mol/dm3 is 0.0091 per second. Conclusions According to the above findings it is true that, from my hypothesis, it is true that, an increase in the amount of the inhibitor has a notable effect on the action of Trypsin enzyme. And that the rates of reactions are much faster without the inhibitor. GRAPH 3 shows an increase in absorbance value as the concentration of mercury nitrate increases. GRAPH 4 shows the lines of best fit for the different concentration of mercury nitrate. Hence the rate of reaction can be got by doing a calculation the gradient of the line. Statistical Test I have decided to carry out a Mann-Whitney U test on the 0.001mol/dm3 and 0.01mol/dm3 concentration of lead nitrate and mercury nitrate.  Rate of reaction when the mercury Nitrate is 0.1mol/dm3 Let’s calculate the rates of reaction when the amount of inhibitor is 0.1mol/dm3 Taking the readings at two different points a (240, 1.57), b (100, 2.0) The gradient/ rate of reaction at that point is =-0.00293 You will realise that the rate of reaction when the concentration of Lead nitrate is 0.1mol/dm3 is -0.00293 per second. Rate of reaction when the mercury Nitrate is 0.001mol/dm3 Let’s calculate the rates of reaction when the amount of inhibitor is 0.1mol/dm3 Taking the readings at two different points a (240, 1.61), b (15,1.74) The gradient/ rate of reaction at that point is =-0.0058 You will realise that the rate of reaction when the concentration of Lead nitrate is 0.1mol/dm3 is -0.0058 per second. Conclusions: From the above findings, the rate of reaction increases with a decrease in the amount of inhibitor used you will realise that, when the amount of inhibitor used is 0.1mol/dm3, the rate of reaction is lower and when amount of Mercury Nitrate is reduced, the rate of reaction increases, the above hypothesis therefore is valid. Limitations of the Mann-Whitney U-Test 1. One major issue with the Mann-whitney’s is that when one fails to get a significant variance in the data under study, one must not conclude that the samples are the similar. 2. Mann- whitney does compare the medians, thus, always do not give a reference to the means found in the conclusion. The above results have shown clearly that, Mercury Nitrate and Lead nitrate do inhibit the rate of reactions. The mechanism through which these two metallic ions does this is that they, combine with the enzyme at the allosteric site on an enzyme hence changing the shape of the active site, the substrate therefore is unable to combine with the active site, therefore the actions of the enzymes gets hindered, hence the reduced rate of reaction, the result of a combination of two,which is the enzyme and the Metal Ion is commonly known as the metal enzyme complex.  Another phenomena that has just been realized is that there exists an inverse correlation, in between the metallic Ions and the enzyme trypsin  Deductions To conclude with, my hypothesis statement has been proved to be true, from the observations made, any slight increase in the, or presence of a metallic Ion in the milk, reduced the rate at which the color of the milk disappears, showing clearly that the rate of reaction of Trypsin is hindered by the presence mercury ions and lead nitrates ions. The rates of reactions therefore are inversely promotional to the amount of lead nitrate solute or the mercury Nitrate used in the experiment. Thus, the higher the amount of the metallic nitrate , the slower rate of reaction, the lower the amount of metallic solute used the faster the rate of reaction  References SMITLEY, W. D. S. (1981). A comparison of the power of the 2independent means u test. Thesis (Ph. D.)--University of South Florida, 1981. NORTHROP, J. H. (1939). Crystalline enzymes; the chemistry of pepsin, trypsin, and bacteriophage. New York, Columbia Univ. Press. LAURITSEN, O. S. (1970). The fibrinolytic enzyme system and trypsin inhibitor as measured by the casein method. Copenhagen, Costers Bogtrykkeri. FAARVANG, H. J. (1965). Urinary trypsin inhibitor in man (mingin); physiological and patho-physiological variations, relation to pituitary-adrenocortical hormones, and to serum trypsin inhibitor. Copenhagen, Munksgaard. BALLESTA, A., STENMAN, U.-H., & TORRE, G. C. (1991). Clinical evaluation of tumor-associated trypsin inhibitor (TATI): proceedings of the symposium in Barcelona on September 24, 1989. Oxford, UK, Published Medisinsk forenings forlag, Uhal by the Black-well Scientific Publications. KALSER, M. H. (1953). Pathophysiology of trypsin, trypsinogen and trypsin inhibitor. Thesis (Ph. D. in physiology)--University of Illinois Chicago Professional Colleges, 1953. ACKERSON, C. W., BORCHERS, R. L., & MUSSEHL, F. E. (1948). Trypsin inhibitor. VII, Comparative nutritive value of raw and heated soybean meal for poults. Lincoln, Neb, University of Nebraska, College of Agriculture, Agricultural Experiment Station. LABO?, G., & VEZZADINI, P. (1980). Serum immunoreactive trypsin: reports from the International Symposium "Advances in the Diagnosis and in the Therapy of Pancreatic Diseases" Bologna, Italy, 30 November 1979. Oslo, Universitetsforlaget. JANEV, R. K. (1991). Collision processes of metallic ions in fusion plasmas: proceedings of the Advisory Meeting of an International A. E. A, Vienna, 16-18 May, 1990. Stockholm, Sweden, Royal Swedish Academy of Sciences. KOMADA, R. (2002). Ions. Digital Video Collection. Princeton, N.Y., Films for Humanities and Sciences. INDIANA UNIVERSITY, BLOOMINGTON. (1944). The Effects of metallic ions and osmotic disturbances on the heart. [Bloomington, Ind.], The University. Read More