Hey! I've attached some background info, could you please answer the questions highlighted in yellow: competing the Effect of pH on the activity of Lactate dehydrogenase enzyme Enzyme activity (moles pyruvate/min), using Beers Law to calculate the rate of reaction as well as the other necessary questions and the two figures. Thank you :)

Introduction Enzymes are proteins that act as catalysts for biological reactions. Enzymes, like all catalysts, speed up reactions without being used up themselves. They do this by lowering the activation energy of a reaction. All biochemical reactions are catalyzed by enzymes. Enzymes are also very specific they only act on one substrate or one class of related substrate molecules. The reason for this is that the active site of the enzyme is complementary to the shape and polarity of the substrate. Lactate dehydrogenase (LDH, L-lactate: NAD oxidoreductase EC 1.1.1.27] catalyses the following reaction: --- CH3 Pyruvate NADH + H+ NAD HO--H lactate dehydrogenase CH3 L-Lactate '= - 25.1 kJ/mol AG' Figure 1. The reaction catalysed by lactate dehydrogenase With this reaction proceeding to the right, the rate of NADH oxidation may be used as a measure of LDH activity. Spectral differences between NADH and NAD* can be used to monitor the oxidation of NADH as shown in Figure 2. 1.0 Oxidized (NAD) 0.8 0.6 EM Absorbance 0.4 Reduced (NADH) 0.2 0.0 220 240 260 280 300 320 340 360 380 Wavelength (nm) Figure 2. Absorption spectra of NAD+ and NADH The absorptivity (extinction coefficient) of NADH at 340 nm is: ENADH = 6.22 x 103 L.mol.'.cm, whilst ENAD+ = 0 L.mol:'.cm1 Therefore, the oxidation of NADH can be continuously monitored by measuring the decrease in absorbance at 340 nm. In this experiment, you will investigate two different parameters that affect enzyme activity: temperature and pH. You will use Beer's Law to calculate the rate of reaction A = ECI Why do you need to use the extinction coefficient for calculations? To analyse the data you are collecting today, you will need to calculate initial velocity, Vo. This initial rate of reaction can be expressed simply as a change in absorbance per unit of time: for NADH consumption this would be AA340/min. This corresponds to the slope on your absorbance vs. time graph. For example, let's say that the straightest portion of your graph is between 0 minute and 1 minute, and the absorbance changes from 0.87 to 0.72 during this time. This means your slope, Vo, (0.87-0.72) is: V = (1-0) Vo = 0.15/min (note that absorbance does not have any units) This is a simple way of calculation. However, it does not allow us to compare the enzyme activity between different reaction conditions. For example, some other enzyme might show the same Vo. Therefore, it is more useful to express the rate in terms of the actual amount of NADH consumed per unit time; this allows researchers working under different experimental conditions to compare their results. Hence, in the current practical, you will calculate the Vo as nanomoles per minute (nmol/min) or micromoles per minute (umol/min). This can be achieved by using the Beer-Lambert law. Make sure you understand this as in the future this can be applied to a wide range of scenarios. We will cover some of this aspect in Lecture 12 - Applications of Biological Chemistry. Effect of pH on LDH enzyme activity In this experiment, you will measure the absorbance every 30 seconds instead of 10 min. Therefore, do each of the reactions independently. Table 2: Assay Mixtures for determining the optimum pH for LDH enzyme activity pH 3 5 6 7.4 8 Citrate buffer 50 mM, pH 3 (mL) 1.8 Citrate buffer - 50 mM, PH 5 (mL) 1.8 Phosphate buffer 50 mM, pH 6 (mL) 1.8 Phosphate buffer 50 mM, pH 7.4 (mL) 1.8 Tris buffer 50 mM, pH 8 (mL) 1.8 Carbonate buffer 50 mM, PH 9 (mL) Pyruvate 3 mm (mL) 1.0 1.0 1.0 1.0 NADH 4.5 mM (mL) 0.1 0.1 0.1 0.1 0.1 Absorbance at zero time LDH - 5 units/mL (mL) 0.1 0.1 0.1 0.1 0.1 1.8 1.0 1.0 Effect of pH on the activity of Lactate dehydrogenase enzyme Table x. table caption pH 3 5 6 7.4 8 9 0.8 0.67 0.87 0.78 0.8 0.75 0.79 0.54 0.71 0.64 0.64 0.52 0.39 0.76 0.48 0.66 0.52 0.47 0.72 0.41 0.58 0.41 0.36 0.21 Absorbance at zero min Absorbance at 30 S Absorbance at 60 s Absorbance at 90 s Absorbance at 120 s Absorbance at 150 S Absorbance at 180 s Aomin - Almin Enzyme activity (umoles pyruvate/min) 0.70 0.36 0.49 0.33 0.09 0.23 0.14 0.69 0.26 0.23 0.05 0.41 0.34 0.68 0.22 0.18 0.09 0.02 CALCULATIONS: show calculations Insert figure Figure x. Absorbance vs. time for all pH Insert figure Figure x. Enzyme activity vs. pH