DifferentialCentrifugationfortheLocalizationofEnzymesandtheExaminationofEnzymeFunction

Differential Centrifugation for the Localization of Enzymes and the Examination of Enzyme Function Sherry Hebert BIOL-3221L-U2020F Alberts, B. (2008). Molecular Biology of the Cell. New York, Garland Science.http://pdpics.com/photo/1865- mushrooms-food/ Lab Procedure Overview ● Differential centrifugation and subcellular localization – Alkaline phosphatase (AP) assay ● Examination of enzyme function – Succinate dehydrogenase (SDH) assay Part A:

Differential Centrifugation ● Use of increasing speed/time of centrifugation to isolate different organelles ● Based on decreasing size/density of organelle of interest Part A:

Differential Centrifugation Centrifuge yeast for 10 min @ 800x g to pellet the cells and discard supernatant Part A:

Differential Centrifugation Resuspend in 5 mL yeast homogenizing solution and transfer to a 50 mL centrifuge tube.

Add 10 mL of glass beads. Part A:

Differential Centrifugation Vortex 30 seconds, on ice 30 seconds for total vortex time of 6 minutes Add 15 mL homogenizing soln Part A:

Differential Centrifugation Strain through 4 layers of cheesecloth, rinsing tube with 5 mL to collect yeast homogenate Part A:

Differential Centrifugation Measure and record the exact volume of yeast homogenate collected.

Collect 1 mL sample for analysis and place on ice. Part A:

Differential Centrifugation Centrifuge homogenate:

15 minutes @ 800x g Post-nuclear supernatant Nuclear pellet Record exact volume of PNS, collect 1 mL sample for analysis (keep on ice) Part A:

Differential Centrifugation Resuspend nuclear pellet in 30 mL homogenizing solution Record exact volume of NP, collect 1 mL sample for analysis (keep on ice) Part A:

Differential Centrifugation Centrifuge post-nuclear supernatant:

30 minutes @ 12,000x g Post- mitochondrial supernatant Mitochondrial pellet Record exact volume of PMS, collect 1 mL sample for analysis (keep on ice) Part A:

Differential Centrifugation Resuspend MP in 5 mL homogenizing solution Record exact volume of MP, collect 1 mL sample for analysis (keep on ice) Part A:

Alkaline Phosphatase Assay Based on production of yellow color when phosphate group is removed from substrate by alkaline phosphatase (AP) p-nitro phenol phosphate (pnpp) AP p-nitro phenol (pnp) P i Part A:

Alkaline Phosphatase Assay Prepare 11 cuvettes containing 2.5 mL of pnpp assay mix + 0.4 mL of distilled water Use arrow on cuvette to ensure proper orientation in spectrophotometer Blank spectrophotometer at 410 nm Part A:

Alkaline Phosphatase Assay Insert cuvette in spectrophotometer and add 100 µ L of yeast homogenate, mix with tip Close lid and record reading = initial absorbance Part A:

Alkaline Phosphatase Assay Keep lid closed and record reading again after 3 minutes = final absorbance Perform duplicate readings using a second cuvette and 100 µ L sample Repeat process for post-nuclear supernatant, nuclear pellet, post- mitochondrial supernatant, and mitochondrial pellet samples Part A:

Alkaline Phosphatase Assay Samples with high AP activity can even be observed visually:

H PNS PMS MPNP Part B:

Examination of SDH Function SDH is a critical component of aerobic respiration:

● Oxidizes succinate to produce fumarate for the TCA cycle ● Transfers electrons to FAD to produce FADH 2 for the ETC Part B:

Examination of SDH Function DCIP is an artificial electron acceptor and can compete with FAD In the presence of azide, which blocks the ETC, all electrons transferred to DCIP by SDH Part B:

Succinate dehydrogenase Assay DCIP ox SDH fumarate succinate DCIP red Part B:

Examination of SDH Function We will determine the effect of altering the components of our assay on the maximum rate of reaction by:

● Altering the amount of enzyme (mitochondrial pellet) ● Omitting DCIP, succinate, or azide ● Adding malonate (a competitive inhibitor) Part B:

Succinate Dehydrogenase Assay Follow the table for the preparation of cuvettes in the lab manual procedure Perform readings at 600 nm: every 5 minutes for 30 minutes in total to determine the maximum rate of reaction We are looking for a decrease in absorbance over time! Data Analysis for Part A Determine the average change in absorbance/min:

● For each replicate determine change by │ absorbance final – absorbance initial │ ● Then average the replicates and divide by 3 minutes Data Analysis for Part A Determine the # enzyme units/100 µ L Divide the average change/min by 0.01 (change in absorbance per µ mol of pnp produced) Data Analysis for Part A Homogenate Post- nuclear supernatant Nuclear pellet Post- mitochondrial supernatant Mitochondrial pellet Units/100 µ l Units/ml Total units % Activity Data Analysis for Part B Create a standard curve for DCIP concentration using the data posted on Nexus ● Don’t forget to force your line through zero! Data Analysis for Part B Create a graph of the absorbance for each sample over time.

● Produce one graph comparing enzyme amount (0.3, 0.6, and 0.9 mL of mitochondrial pellet) ● Produce another graph for all tubes with 0.6 mL of mitochondrial pellet Data Analysis for Part B Determine the 5 minute interval with the greatest change in absorbance for each sample.

Max rate DCIP reduction = ( Δ absorbance / 5 minutes) slope From DCIP standard curve Assignment ● Is available in the Lab 3 Nexus folder ● Is due by 11:59 PM on October 22 nd and is worth 2% of your final grade ● Includes calculations, formal table & figure prep, and answering questions related to the theory and analysis of your data