Introduction In this lab, we will investigate the kinetics of LDH purified from muscle in order to learn how to determine

kinetic information by plotting kinetic data. The plots we will use for this lab is the Michaelis-Menten Plots and the Lineweaver-Burk Plots. By plotting the kinetic data, we will solve and understand the effects of inhibitors on enzyme activity. Discussion For this lab, the numbers that I originally obtained from the experiment using my own LDH (shown on page 68 70) were not adequate enough for me to do perform analysis on them. First, my LDH had a dilution of 100 and the activity for it was still 0.015, which did not even make the 0.02-0.04 range. Comparing the LDH activity from this week to last week, it is apparent that my activity since last week has decreased a tremendous amount. This could be because the LDH was not kept on ice all the time and therefore lost activity. It could also be because the LDH concentrated fraction had very low activity to begin with (in Chapter 3, I only had 41.506 units/mL), most likely due to an error during the affinity chromatography stage in Chapter 3. Since my activity was so low, I got very low values for the kinetics in this lab (as seen on page 68-70). Therefore, I obtained TF data from Alex and Cody (copied on page 72). For the analysis, instead of using my data, I used the TF data (experiment done by D-A). Although I believed the calculated value of KI is correct, I feel that the value is too high. However, after comparing with part 5 of the Notebook, where I compared the Km and Vmax values obtained through the Michaelis-Menten Plot, I found that the values for Km and Vmax (that I calculated with the Lineweaver-Burk Plot) are around where they are supposed to be. Also, there cannot be much error on the regression line because the R2 value is 0.9978 for the inhibited and 0.9863 for the uninhibited line, which show very strong linear characteristics. For determining the type of inhibition, the Lineweaver-Burk Plot was very hard to follow. Although the two lines seemed to intersect, when looking at their regression line slopes, the two seemed very similar (was off by 0.0004). Therefore, I believed that this inhibition was most likely uncompetitive inhibition. A better way to see if it actually is the uncompetitive inhibition would be to take more sample points so that the data would have more data points to make a linear regression off of. That way, it would be clear whether the slopes were actually different or were supposed to be the same. Conclusion This lab was very important in teaching me what plot was useful for what type of circumstance. The Lineweaver-Burk Plot is very easy to understand and also very easy to construct. However, the Michaelis-Menten Plot is a very good source to check ones values that they obtain through calculation with the Lineweaver-Burk Plot. It is especially good when the values you obtain through the LB plot seem very off. This type of analysis is very important because studying inhibitors in protein is a large part of lab in the real world. Biochemists and the like have to deal with inhibitors every day, in order to determine whether a certain ligand inhibits a biochemical reaction and how that affects the physiology of the human body.