Chapter 5: Structural Characteriza1on of LDH Purpose of Week 3: C) Separate isozymes of LDH by charge via na6ve zone electrophoresis (zymography)! Load 1S crude extract + heart & muscle isozyme standards onto cellulose- acetate membrane! Run electrophoresis! Detect separated bands of dis?nct isozymes using ac?vity staining technique
Clinical significance of isozyme detec1on Useful for diagnosis of various condi6ons involving 6ssue damage & other disease states metabolic disorders myocardial infarc6on organ damage
Zymogram overview Can be used to separate enzyme molecules that catalyze the same reac6on but differ in charge due to slight varia6ons in their amino acid sequences (isoenzymes or isozymes) + direc1on of electric current cellulose- acetate membrane: Origin (where samples are loaded) (- ) charged isozymes mobility (+) charged isozymes
Separa1on of isozymes by electrophoresis Samples are applied to cellulose- acetate membrane (soaked in buffer) Current applied to membrane Isozymes separate according to net charge Direc6on & speed of migra6on is ph dependent Process does not denature proteins Enzymes retain ac;vity
Ac1vity staining allows visualiza1on of separated isozymes Immediately aier electrophoresis, membrane is applied to an ac1vity gel assay plate The following rxns will occur & stain where LDH is on the membrane:
Isozymes of LDH Homo- and heterotetrameric LDH isozymes found in the body Composed of 1ssue- specific combina6ons of H (heart) &/or M (muscle) subunits Subunit variants: M 4, HM 3, H 2 M 2, H 3 M, H 4! 5 isozymes total based on quaternary structure Isozymes have same func6on & overall 4 o structure with slight differences in kine6c parameters, 1 o structure & net charge
Example LDH zymogram Figure 5-21 from page 169: + Heart standard 1S crude - heart Muscle standard 1S crude - muscle Isozymes in order of migra6on: M 4, HM 3, H 2 M 2, H 3 M, H 4 Dean R. Tolan, 2012
Chapter 5C Procedure Workflow for Chapter 5 week 3: Prepare samples Prepare membrane and applicator Apply samples to membrane Run zymogram Prepare ac6vity staining gel (while running zymogram) Stain zymogram Image zymograms (and Coomassie- stained SDS- PAGE if needed)
Chapter 5C Procedure Become familiar with applicator apparatuses (h\ps://www.youtube.com/watch?v=ihff51wmm5k) Stowed applicator Applicator cover (with water reservoir) Applicator base (with sample slots) Applicator Applicator base (no1ce the numbering of the sample slots)
Prepare samples Chapter 5C Procedure You will need 20 μl of 1S containing 1 to 2 U of LDH Calculate the [Ac6vity] of your 1S aliquot If <50 U/mL, consult TF If between 50-100 U/mL, use 20 μl of your sample directly If significantly greater than 100 U/mL, need to dilute your sample: o Calculate volume needed for 100 U of ac1vity o Aliquot into a new tube and adjust volume to 1 ml with EDB o This is your diluted zymogram sample (use 20 μl) Keep samples & standards ON ICE un;l ready to load You will use your 1S aliquot and ac6vity data from Chapter 3 to prepare your sample for this week You will be paired with a group that has the opposite 6ssue isozyme (2 groups / zymogram; 1 heart & 1 muscle, no excep;ons) If you are the odd group, ask another group for sample of opposite 1ssue isozyme and make sure you have both standards Each zymogram must be complete for a thorough post- lab analysis
Chapter 5C Procedure Prepare membrane and applicator Do not begin un;l both of your groups samples have been prepared Your TFs have pre- soaked the cellulose- acetate membranes in barbitol buffer Cau1on! Barbitol is poisonous. Handle membrane with forceps. If you touch the barbitol buffer, throw gloves into hazardous waste Secure soaked membrane on plas6c bridge WORK QUICKLY! DO NOT LET THE MEMBRANE DRY OUT!!! Wash applicator keys 3x in dih 2 O in the cover reservoir, and test on a paper towel (h\ps://www.youtube.com/watch?v=wbmdrcuew5q) Apply samples to membrane Pipet samples & standards onto wells of applicator base (pay close ahen?on to number order [#4 on lel, #1 on right]) Place applicator on base, hold down bu\on for 1 second, transfer applicator to bridge, hold down bu\on for 1 second to apply samples (h\ps://www.youtube.com/watch?v=m5jrjmx_yu8)
Chapter 5C Procedure Run zymogram Do not manipulate electrophoresis chamber without TF supervision Place loaded zymogram membrane bridge inside of electrophoresis chamber Pay azen;on to which side is marked + vs. - Run your zymogram at 200V for 45 min Prepare ac1vity staining gel (while zymogram runs) In a 15 ml conical tube, add these reagents in the following order:! 3.2 ml lactate! 4.0 ml NAD+! 0.96 ml phenazine methosulfate (add dropwise)! 1.2 ml tetranitroblue tetrazolium *Cau;on! (carcinogenic!) Quickly transfer 16 ml of molten Nobel agar into 50 ml conical tube Pour ~10 ml of reagent mix into Nobel agar, invert several 6mes, and pour into a 15 cm petri dish. Carefully cover dish in foil (light- sensi6ve reagents) & place in fridge Do not touch for 15 min otherwise gel won t polymerize properly.
Chapter 5C Procedure Staining your zymogram Remove membrane from bridge & place topside facing down onto the polymerized agar Carefully place assay plate in 37 C incubator for 10 min to develop color Image zymogram under TF supervision using gel doc in back room
Chapter 5 Week 3: Before the lab period, you should have: " Completed your prelab " Title, date, introduc6on, procedures " Include volume calcula6on for Units (U) of 1S crude sample to load if [Ac6vity] > 100 U/mL At the end of lab, you should have: " Ran zymogram " Ac6vity stained & imaged zymogram " Imaged your SDS- PAGE from last week (if did not finish)
Notebook calcula1on of k cat from Ch. 3-5 Data Use V max from Chapter 4: Units/mL = μmol/(ml*min) = mm/min Example: V max = 200 Units/mL = 200 mm/min Use protein concentra1on from Chapter 3: mg/ml of protein = g/l of protein Example: 50 mg/ml of protein = 50 g/l of protein Mul1ply protein concentra1on by 1/Subunit M r from Chapter 5: Note that M r in Da = g/mol (g/l of protein)(1 mol/g of LDH)(1000 mmol/1 mol) = mm of protein Example: [E T ] = (50 g/l)(1 mol/25,000 g of LDH)(1000 mmol/1 mol) = 2 mm of protein Final Answer: k cat = V max /[E T ] =(mm/min)/(mm) = 1/min k cat = (200 mm/min)/(2 mm) = 100/min # convert to sec - 1 Turnover Number = k cat = V max /[E T ]
Es1ma1on of isozyme % from LDH zymogram (Part C.2. in Notebook) + Heart standard 1S crude - heart Muscle standard 1S crude - muscle Es?mate % of M 4, HM 3, H 2 M 2, H 3 M, &/or H 4 isozyme present in samples based on band intensi;es (Total has to add up to 100% for each sample!) Dean R. Tolan, 2012
Ques1ons?