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<p class=”(1)”></p> | <p class=”(1)”></p> | ||
</div> | </div> | ||
− | <p class="content">Where, | + | <p class="content">Where, [<i>P </i>] is the concentation of Pantide, <i>K<sub>h</sub></i> is the reaction constant of hydrolysis, and <i>k<sub>A</sub></i> , <i>k<sub>N</sub></i> , <i>k<sub>B</sub></i> are the three contributing components of overall hydorlysis which are dependent to condition.</p> |
− | + | <p class="content"> For Pantide, we would apply the protein solution in the farm at a constant pH. Thus, the rate of hydrolysis is proportional to the concentration of Pantide by a constant <i>K<sub>h</sub></i> , and the concentration of Pantide undergoes an exponential decay as time goes on.</p> | |
− | <p class="content"> For Pantide, we would apply the protein solution in the farm at a constant pH. Thus, the rate of hydrolysis is proportional to the concentration of Pantide by a constant <i>K<sub>h</sub></i>, and the concentration of Pantide undergoes an exponential decay as time goes on.</p> | + | |
<p class="content-1" style="color:#00E600"> ii. Experimental proof</p> | <p class="content-1" style="color:#00E600"> ii. Experimental proof</p> | ||
<p class="content">We tested the chemical stability of both native and linear types of Hv1a and Hv1a-lectin in neutral PBS solvent (phosphate buffered saline, pH=7.4) in 4 ℃ for one day and seven days. SDS-PAGE showed the remained protein concentration and calculated by software ImageJ. (Figure 2, Figure 3)</p> | <p class="content">We tested the chemical stability of both native and linear types of Hv1a and Hv1a-lectin in neutral PBS solvent (phosphate buffered saline, pH=7.4) in 4 ℃ for one day and seven days. SDS-PAGE showed the remained protein concentration and calculated by software ImageJ. (Figure 2, Figure 3)</p> | ||
− | <p class="content"></p> | + | <div> |
− | <p class="content"></p> | + | <img src=”” class=”picture”> |
+ | <p class=”Figure 2. SDS-PAGE gel and the concentrations of hydrolysis test to Hv1a (5.3 kDa). The samples were marked on the top of the gel.”></p> | ||
+ | </div> | ||
+ | <div> | ||
+ | <img src=”” class=”picture”> | ||
+ | <p class=”Figure 3. SDS-PAGE gel and the concentrations of hydrolysis test to Hv1a-lectin (HL, 17.1 kDa). The samples were marked on the top of the gel.”></p> | ||
+ | </div> | ||
+ | <p class="content">The difference of the concentration of native Hv1a was not significant during seven days (1 day: 98%, seven days: 87%), while linear Hv1a only remained 9% after one day, and almost all degraded after seven days.</p> | ||
+ | <p class="content">The test to Hv1a-lectin showed the similar result. Native Hv1a-lectin nearly did not degrade in 7 days (1 day: 110% , 7 days: 105%), but linear Hv1a-lectin remained 16% after 1 day, 3% after 7 days.</p> | ||
+ | <p class="content">So, we could conclude that native proteins dissolved in neutral PBS solvent did not undergo hydrolysis (or at a very slow rate) in 4 ℃ for seven days, on the other side, linear form proteins degrade as time went on, and remained merely little after seven days.</p> | ||
</div> | </div> | ||
</div> | </div> | ||
+ | |||
+ | |||
<!--4--> | <!--4--> | ||
<div class="modelingPart"> | <div class="modelingPart"> | ||
<h2 class="content-1" id="titleO" style="color:#FFFF82">IV. Proteolysis test</h2> | <h2 class="content-1" id="titleO" style="color:#FFFF82">IV. Proteolysis test</h2> | ||
<div class="modelingPartContent" id="partO"> | <div class="modelingPartContent" id="partO"> | ||
− | <p class="content"></p> | + | <p class="content-1" style="color:#00E600"> i. Theory</p> |
+ | <p class="content">To simulate the degradation by protease, we used Michaelis-Menten kinetics model to express the rate equation. It is useful to describe enzymatic reactions, especially the one without ligand participation, such as proteolysis with proteases whose mechanism is first to find out the specific active site and then react to break it down. [3]</p> | ||
+ | <p class="content">The proteolysis process is irreversible in vivo because the product, linear form protein, is degraded quickly. We used differential equation to describe the major proteolysis process:</p> | ||
+ | <div> | ||
+ | <img src=”” class=”picture”> | ||
+ | <p class=”(2)”></p> | ||
+ | </div> | ||
+ | <p class="content">Where [<i>P </i>]: the concentation of Pantide, <i>V<sub>m,p </sub></i> : the maximum reaction rate of proteolysis, which is equal to the product of the concentration of total enzyme and turnover number <i>k<sub>cat </sub></i> of specific protease and substrate, <i>K<sub>M,p </sub></i>: Michaelis constant, which is the substrate concentration at which the reaction rate is half of <i>V<sub>m,p </sub></i></p> | ||
+ | <p class="content">We had assumed that the <i>V<sub>m,p </sub></i> is a constant because the proteases in vivo are in active and in equilibrium, and <i>V<sub>m,p </sub></i> represents the equivalent value for all kinds of protease in environment.</p> | ||
+ | <p class="content-1" style="color:#00E600"> ii. Experimental proof</p> | ||
+ | <p class="content">We designed two experiments to test the enzymatic stability towards protease of Hv1a and Hv1a-lectin. One was to observe the degrade level of both native and linear types of proteins applied by protease for one day, and the other one was to obtain the curve of the degradation rate of only linear form proteins in the period of four hours, because of the resistance against the protease.</p> | ||
+ | <p class="content">For the first experiment, we dissolved the protein solutions in neutral PBS (pH=7.5) solvent, applied with 0.25% trypsin-EDTA(1:250), a serine protease, and then incubated the samples in work temperature 37℃ for one day. (Figure 4, Figure 5)</p> | ||
+ | <div> | ||
+ | <img src=”” class=”picture”> | ||
+ | <p class=”Figure 4. SDS-PAGE gel and the concentrations of trypsin resistance test to Hv1a (5.3 kDa). The samples were marked on the top of the gel.”></p> | ||
+ | </div> | ||
+ | <div> | ||
+ | <img src=”” class=”picture”> | ||
+ | <p class=”Figure 5. SDS-PAGE gel and the concentrations of trypsin resistance test to Hv1a-lectin (HL, 17.1 kDa). The samples were marked on the top of the gel.”></p> | ||
+ | </div> | ||
+ | <p class="content">Compared with the sample incubated for one day without trypsin treatment, native Hv1a and Hv1a-lectin showed the resistance to trypsin protease (111% and 100%), but linear proteins were degraded by proteolysis (67% and 18%).</p> | ||
+ | <p class="content">We next tested the proteolysis rate of only linear proteins in the period of four hours, and we drew the Lineweaver–Burk plot (also called “double reciprocal plot”) (Figure 6) to obtain <i>V<sub>m,p </sub></i> and <i>K<sub>M,p </sub></i> for linear form Hv1a and Hv1a-lectin. (Table 1)</p> | ||
+ | <div> | ||
+ | <img src=”” class=”picture”> | ||
+ | <p class=”Figure 6. Lineweaver–Burk plot of proteolysis test of linear Hv1a (blue line) and Hv1a-lectin (orange line). The horizontal axis represents the reciprocal of substrate concentration, and the longitudinal axis represents the reciprocal of rate. The straight line’s x-intercept means the reciprocal of -<i>K<sub>M,p </sub></i>, and y-intercept means the reciprocal of <i>V<sub>m,p </sub></i>.”></p> | ||
+ | </div> | ||
+ | <div> | ||
+ | <img src=”” class=”picture”> | ||
+ | <p class=”Table 1. The<i>V<sub>m,p </sub></i> and <i>K<sub>M,p </sub></i> of linear form Hv1a and Hv1a-lectin.”></p> | ||
+ | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
+ | |||
+ | |||
<!-5--> | <!-5--> | ||
<div class="modelingPart"> | <div class="modelingPart"> | ||
<h2 class="content-1" id="titleP" style="color:#FFFF82">V. UV radiolytic oxidation test</h2> | <h2 class="content-1" id="titleP" style="color:#FFFF82">V. UV radiolytic oxidation test</h2> | ||
<div class="modelingPartContent" id="partP"> | <div class="modelingPartContent" id="partP"> | ||
+ | <p class="content-1" style="color:#00E600"> i. Theory</p> | ||
+ | <p class="content">In nature, proteins are also probable to degrade under the sunlight, and the reason is that the energy of light excites the solvent (almost water) and initially generate radicals, which afterward propagate and attack protein to break it down until termination.</p> | ||
+ | <p class="content">Ionizing radiation causes the radiolysis of water is, the major process is shown below. (Figure 7)</p> | ||
+ | <div> | ||
+ | <img src=”” class=”picture”> | ||
+ | <p class=”Figure 7. The radiolysis process of water.”></p> | ||
+ | </div> | ||
+ | <p class="content">Though the real mechanism that radicals attack to protein is quite complex, we can simply indicate the rate of protein been attacked by an unknown power, n, of the total concentration of radicals. Then we derived the rate formula as differential equations.</p> | ||
+ | <div> | ||
+ | <img src=”” class=”picture”> | ||
+ | <p class=”(3)”></p> | ||
+ | </div> | ||
+ | <div> | ||
+ | <img src=”” class=”picture”> | ||
+ | <p class=”(4)”></p> | ||
+ | </div> | ||
+ | <p class="content"> Where [<i>radical </i>]: the total concentation of radicals, [<i>P </i>]: the concentation of Pantide, <i>G<sub>γ </sub></i>: the dose rate of absorbing γ-ray (radiation energy absorption rate per mass, for water, 1.42 Gy/s) [5], <i>A<sub>γ </sub></i>: the number of radicals created per energy (for water, 0.045 μmol/J) [5], <i>I </i>: the intensity of UVB from sunlight measured by UV Sensor (UVM30A), <i>R<sub>T </sub></i>: the rate constant of radicals termination, which is equal to 2.365×10<sup>-7 </sup>mol<sup>-1 </sup>s<sup>-1 </sup> [6], <i>K<sub>UV </sub><i>: the rate const ant of UV radiolytic oxidation to protiens, which is set to 44 </sup>mol<sup>-1 </sup>s<sup>-1 </sup>at the beginning [6]</p> | ||
+ | <p class="content">We then used software MATLAB to simulate the degradation rate by UV radiolytic oxidation on the intensity of UVB from sunlight. The results showed that the degradation rate increases as rising intensity whatever n is, and eventually it tends to be fully degraded. (Figure 8)</p> | ||
+ | <div> | ||
+ | <img src=”” class=”picture”> | ||
+ | <p class=”Figure 8. The simulation of degradation rate by UV radiolytic oxidation on the intensity of UVB from sunlight.”></p> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p class="content-1" style="color:#00E600"> ii. Experimental proof</p> | ||
+ | <p class="content"></p> | ||
+ | <p class="content"></p> | ||
+ | <p class="content"></p> | ||
<p class="content"></p> | <p class="content"></p> | ||
</div> | </div> |
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