Lab 3 Questions Question B-1.

It seems like the largest difference is at 340nm, where NADH reaches its peak and NAD is a flat line. This is because NAD+ has no absorbance at 340nm. In order for absorbance to show at 340nm, the amount of energy from the absorbance of light at 340nm needs to promote the lone pair of electrons on the nitrogen of the pi-bonding orbital to the lowest unoccupied orbital in the structure, which is a pi* orbital. Therefore, it takes a lot more energy to move the lone pair of electrons on the nitrogen of NAD+ than what the wavelength at 340nm gives off. In contrast, the lone pair of electrons on NADH is a nonbonding orbital on the dihydronicotinamide ring, and can be promoted at a much lower energy level (lower wavelength) than the lone pair of NAD+. Therefore, NADH is able to show absorbance at 340nm because it requires less energy to move an electron to its lowest unoccupied orbital.

Question C-1. The rate of decrease of the LDH reaction seems to stay constant with time. The graph had a constant slope of -0.0013 with an R2 value of 0.99988, which says that the decrease in absorbance was constant and closely fits the linear model. This shows that as time went on, the A340 decreased constantly and therefore gives the LDH reaction a constant decrease with time. However, this is only for one part of the entire reaction. The rate of LDH reaction starts out as a linear decline, but eventually the rate decreases with time and then becomes a flat line. This is because there are three parts to the rate of the LDH reaction vs time graph. In the first part, the slope is a constant like is shown in Graph 2. This is because in the reaction, there is full saturation of NADH, which the enzyme hits continuously until all enzyme is used. In the second part, the slope starts to curve and the rate starts to slow down because the reaction is at partial saturation, where some of the substrate already formed the product, and the available substrate continues to bind with the enzyme. Since there are already other particles in the solution (reaction system), it becomes harder for the enzyme to hit the substrate and thus deceases the reaction rate. In the third part, the slope completely flat lines because at this point all the substrate (NADH in this case) has gone to completion so there is no more substrate to make product. Question C-2. In our graph, the LDH reaction increased with an increase in enzyme concentration. However, if the enzyme concentration kept increasing, the graph would have a linear increase in the beginning and then it would hit an asymptotic curve when it reaches a certain point. This is because in the beginning, the limiting factor is the amount of enzyme there is in the reaction, so the linear line suggests a direct relationship between the enzyme concentration and the rate of reaction. In the second part of the graph, the change of the rate (the change of the A340/min) begins to decrease. This happens because in the second part, the increased amount of enzyme is no long the limiting factor. In this case, both the enzyme concentration and the substrate concentration approach an equal concentration, and therefore this slows the change in reaction rate because there is only so much substrate in the reaction to start with. In the third part of the graph, the line flat lines, and this is because the limiting factor becomes the substrate and the reaction happens too fast because of the high enzyme concentration. Question C-3. The rates tend to decrease after an extended reaction time because the substrate NADH (since that is what we are measuring) gets used up and turned into product, NAD+. If the enzyme concentration is high, that means that the reaction happens faster and therefore product production also happens faster. That is why on the A340/min kinetic graph on the spectrophotometers, we would only see a flat line when our enzyme concentration was too high, because the reaction happened so fast that the spectrometer was not able to measure the decrease in NADH fast enough. Question C-4. The number of units of LDH per gram tissue is 19.62units/mg for 1S, 22.43units/mg for 2S, 0units/mg for 2P, 2.78units/mg for 3S, 9.39units/mg for 3PD, 41.81units/mg for 3PD, 131.6units/mg for the pooled fractions and 0units/mg for the concentrated.

Question D-1. During dialysis, the volume did change because while the salts in the original semi permeable membrane bag moved out of the bag to reach equilibrium with the outside, (meaning that it increased the salt concentration outside, making the ammonium sulfate concentrations inside and outside the bag equal) the bag still contains large enzyme molecules that cannot go through the membrane. This allows for water to go into the bag because it tries to decrease the enzyme concentration by increasing the total volume of solution. Therefore, the reason that the volume increases is because the system tries to reach equilibrium by increasing volume and thus decreasing the enzyme concentration in the bag. Question D-2. When you put salt into water, the salts dissociate because the water molecules pull the ionic bonds apart, since they have such a high affinity for them (because of the strong positive charge pulled by the negative charge on the oxygen of water molecules). Because of this characteristic, when you have a low concentration of a salt, like Ammonium sulfate, in solution, it can help stabilize the solubility of protein, since one of the factors that influence protein solubility is the salt concentration in the solution. However, as you increase the salt concentration, as we did in lab by adding the 19.7ml of Ammonium sulfate, it starts to take most of the water molecules for itself, which leaves less water molecules to interact with the protein molecules. As you increase the Ammonium sulfate concentration, the amount of water molecules available for the proteins decrease, and eventually, the protein will precipitate because it gets excluded from the water molecule binding with Ammonium sulfate. Question D-3. Proteins are usually least soluble at their isoelectric pH, which is the pH at which their net charge is 0. For proteins, different pH conditions can result in different packing orientations due to the structural change in protein. At the isoelectric point, since the net charge of the protein is 0, it becomes easier for the proteins to stack together. This packing together of protein makes it hard for other solutes such as Ammonium sulfate or even water molecules to have a strong interaction with the protein molecules. Therefore, at the isoelectric point, proteins can precipitate out. Another reason why the pH can have an effect on protein crystallization is the way it changes the charge orientation on water molecules. For water molecules, it is easy for the positively charged hydrogen atoms to align themselves along a negatively charged protein molecule and create a wall of hydrogen atoms. In order to increase entropy for the water molecules in solution, the water molecules will eventually push all the protein molecules together so that the surface area that the water molecules will have to surround will decrease, thus increasing entropy of the free water molecules that will not have to surround the protein molecules. This mechanism is called clathrate structures of water.

Introduction The purpose of this lab is to do a purification of Lactate Dehydrogenase (LDH) using multiple steps while measuring the kinetic assays for each step. In the first week, we will learn the basic techniques for protein purification, prepare a crude extract by centrifugation, and then run enzyme assays to determine the amount of enzyme there is in the sample by measuring the amount of NADH in the sample with time. In the second week, we will learn and understand the effects of ionic strength on proteins and precipitate proteins using ammonium sulfate, and then use dialysis to de-salt a protein sample. In the third week, we will use affinity columns to purify proteins, learn how to actually make an affinity column and to use ultrafiltration to concentrate the protein samples after we take fractions of them through the affinity column. All these steps eventually lead to a concentrated purified LDH sample. Conclusion The Bradford Assay is still the simplest way to measure the amount of protein you have in a sample. Although you have to assume that the amount of LDH shows up in a similar amount to the amount of BSA used to construct the standard curve, the Dye-Binding Assay is a quick and simple technique, which therefore means that there is less room for error. Also, the standard curve can be improved by increasing the number of data points you take for the curve. This will also allow you to get a better fit line and you would be able to increase the limits on the standard curve. This lab allowed me to understand the mechanism of protein purification in particular, the affinity column. The affinity column had AMP bound to the surface, to which the LDH would bind because of its strong affinity to AMP. By eluting the column with NADH, the AMP-bound LDH lets go of the AMP and binds to NADH because it binds more tightly than it does to AMP. This type of technique is used frequently in lab, in the form of techniques such as ELISAs, which stand for Enzyme-linked Immunoabsorbent Assays. ELISAs are similar to affinity columns (the binding part is, at least) but instead of binding enzymes with substrates, you have enzymes bind with antibodies and antigens in order to measure the concentration. In conclusion, this lab was important because it allowed me to understand the importance of protein purification, since protein purification is the most important aspect in an experiment when someone discovers a new protein.

Discussion All in all, the results that I obtained for this lab were reasonable up until the pooled sample. This is apparent because when you compare the concentration of protein of the assays (which is supposed to increase as you move on in the purification because you would have more protein in the same amount of volume used to do the kinetics and the Bradford Assays) you notice that in the beginning, it starts off as 109.9mg/ml. It keeps increasing (except for the assays that do not have much protein in them such as 2P and 3S), until it reaches its maximum at the 3P. The 3PD has a lower concentration of protein than the 3P fraction because during dialysis, the total volume increased because of net water movement into the dialysis bag to bring to equilibrium the concentration of protein molecules. After the 3PD, however, the concentration of protein dropped 100-fold. This probably means that our error was in not choosing the correct fractions for the pooled fraction. In lab, while we were doing the kinetics for the fractions, we found that fraction #4, 5, and 6 showed enzyme activity (shown in Graph 4). Although the activity was low, we believed from the pattern of enzyme activity of the fractions (as in fraction 3 showed no activity, fraction 4 showed some activity, fraction 5 showed more activity than 4, fraction 6 showed less activity than 5 and fraction 7 showed no activity all shown in Graph 4) that fractions 4, 5, and 6 were the ones that had the most of the enzyme in them. However, since the concentration of protein of the pooled had such a low value (1.626mg/ml), this means that the fractions we pooled probably did not contain most of the LDH we were purifying. This also causes another problem in the Purification Table. For the Concentration of Protein of 2P, 3S, Pooled and Concentrated, the values of A595 obtained for the DyeBinding curve were not between the values of 0.229~1.146, which is the region where the standard curve is a linear fit. However, before and after the points of 0.229 and 1.146 respectively, because you dont know how the curve actually looks like (because it could look like an exponential curve or it could look like an asymptotic curve), the calculations using the same linear fit equation for values under 0.229 or over 1.146 have a much larger margin of error than those who are between the values. For the purpose of calculation for the purification table, however, I used the best-fit line of the dye-binding standard curve to calculate the concentration of protein, to get an estimate. This is also why the concentration of the 2P and concentrated fraction is a negative value, since their absorbance values were below the y-axis of the standard curve, which was 0.089. While, we should have done the dye-binding for the pooled and concentrated over with a higher concentration (we had a 1:50 dilution), we would have run out of the concentrated fraction because we had a low pooled fraction to start with. Perhaps, if we had more time, a good way to make sure that our pooled concentration is that value (1.626mg/ml) or not would be to do the kinetic assays for the rest of the fractions (if we still had them). If more enzyme activity shows up in the later fractions (after fraction 7), which would mean that we made an error in packing the column (since we made an error the first time through and the second time we accidently dried the column although it was all before we ran the 9ml of 3PD).