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UB BIO 201 - Lab 4

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Lab 41. What enzyme are we analyzing, what reaction does it catalyze, where does it exist naturally, and what is our source of enzyme for this lab? - Lactase- Lactase catalyzes the hydrolysis of lactose to galactose and glucose.- Gastrointestinal tracts- Lactaid tablets2. What is an extinction coefficient? The extinction coefficient is a measure of how much light a 1M solution absorbs at a specific wavelength and is a constant for a given spectrophotometer.3. Which characteristic of a chromophore is directly proportional to concentration: absorbance or transmittance? Absorbance.4. If a chromophore has a transmittance of 10% (i.e., 0.1) at 480 nm, what is its absorbance at 480 nm? A = -log T = -log 0.1 = 1 5. In Lab 5 we will be using the chromophore DCPIP. If its extinction coefficient is 21,000 at600 nm, calculate the A600 of a solution of 0.01 mM DCPIP, assuming path length is 1 cm? A = lc = 21,000Lmol cm  1 cm x 0.01mmolL  mol1000 mmol=¿0.216. What is the concentration of a solution of DCPIP that has an A600 of 0.21? c = A/l =0.2121,000Lmol∗cm∗1 cm = 0.00001 M (If you convert to mM, this is 0.01 mM.)7. Why are enzymes more sensitive to the effects of pH than most other proteins? Because even a slight change in pH can alter the conformation and chemistry of the active site on the enzymes. Although the mechanisms the enzymes use to catalyze reaction might be different, but all involve a specific binding interaction between the substrate and the enzyme’s active site.8. In Part 1 of the lab, why do you need to test different dilutions of enzyme? To determine a concentration of lactase that the absorbance is high enough to be readable, but not so high that it maxes out the spectrophotometer. 9. State an experimental hypothesis for Part 2 of this lab. The pH optimum of lactase is ______.Lmol cm10. What are the independent and dependent variables in Part 2 of this lab? Independent variable: pHDependent variable: absorbance (or enzyme activity)11. What is the purpose of the ONPG in this lab (be specific)? ONPG is a false substrate for lactase. When active, lactase hydrolyzes ONPG to galactose and o-Nitrophenol. o-Nitrophenol spontaneously ionizes to the o-Nitrophenolate ion, which is yellow. Therefore, the activity of lactase can be studied by measuring the increase in formation of yellow product using spectrophotometry.12. What new equipment will you be using in this lab? Serological pipets, pipet pump, spectrophotometer.13. What are the safety hazards in this lab? Glass pipets,


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UB BIO 201 - Lab 4

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