University:
Boston University
Course:
CAS CH 101 | General Chemistry 1
Academic year:

2024
Views:

48

Pages:

17
Author:

hieti9j8

Purification of an Enzyme Biochemical Isolation of Lactate Dehydrogenase (LDH) from bovine tissue Overall goals of this chapter:   Learn the step‐wise purification techniques for isolating an enzyme Analysis of enzyme activity & yield Lactate Dehydrogenase (LDH) ● ● LDH catalyzes the last step of anaerobic glycolysis Multiple forms of LDH found in different tissues – Isozymes ● Each isozyme has slightly different kinetic and structural properties, but same function and overall structure Ch. 3 Protein Purification Process Disruption of tissue • Homogenization achieved by blending minced tissue + buffer (Cell lysis) • Separation via centrifugation • Pellet – Cell Debris • Supernatant – Cell Extract (lysate) Clearing the cell lysate Removal of additional contaminants Isolation of LDH • Protein precipitation via • Further separation by affinity chromatography Purified Protein The TWO MOST IMPORTANT FACTORS for Chapter 3 success: 1) Maintaining HIGH ENZYME ACTIVITY *Keep your enzyme COLD & AVOID BUBBLES!!! 2) Maximum recovery of protein (HIGH YIELD) *Don’t be sloppy… Do the procedures correctly. *PAY ATTENTION to what needs to be saved each week!!! Comportment grade penalties for throwing out critical fractions! Why is Chapter 3 such a big deal?? Top 5 reasons: 5) Notebook write‐up is worth A LOT… 3x CH1 pts 4) The notebook write‐up will take FOREVER. 3) Enzyme purification is DIFFICULT. [effort + attention = insanely critical] 2) Will need your purified LDH for Ch 4 & 5 too. 1) Increasing SPECIFIC ACTIVITY is your main focus. *Should go up with each purification step. Low/no activity = sad/useless enzyme  Objectives for Chapter 3 Week 1: A) Prepare a crude enzyme extract from bovine heart or skeletal muscle *Note: You will be assigned heart or muscle by your TFs! B) Obtain spectra of NAD+ and NADH to determine εapp of NADH C) Assay for LDH activity of crude extract *Mastery of enzyme activity assay is critical for Chapters #3‐5!!! Part A: Preparation of Crude Extract Steps: 1) Homogenize Homogenize beef beef sample sample 2) Centrifuge to clear lysate 3) Collect crude extract + K/PO4 buffer + Part A: Preparation of Crude Extract Steps: 1) Homogenize beef sample 2) Centrifuge to clear lysate 3) Collect crude extract Purpose of Centrifugation  Uses centrifugal force to separate sample components  Separates lysate from cell debris Homogenate (“Meat smoothie”) Centrifuged sample 1S crude extract High speed Collect cell lysate centrifugation Discard pellet Cell debris, organelles LDH Carbohydrates and other small molecules Extraneous proteins and other macromolecules Part A: Preparation of Crude Extract Steps: 1) Homogenize beef sample 2) Centrifuge to clear lysate 3) Collect crude extract (contains fat deposits) 1S supernatant (collected) Centrifuge tubes: 1P Pellet (discarded) After Step 2 centrifugation… ‐ Pour centrifuged 1S sample through cheesecloth ‐ Collect filtered 1S sample Your TFs may advise you to skip the cheesecloth step if your centrifuged 1S sample is clean and free of fat Tips: Preparation of Crude Extract • Before blending, weigh & record mass of initial beef sample = ? g *TFs will have already pre‐weighed & minced approximate amounts for you • After blending, record total volume of homogenate = ? mL • After cheesecloth, record volume of filtered crude 1S sample = ? mL Tips: Preparation of Crude Extract (cont’d) • Before centrifugation, balance tubes & record total volume that went into the tubes **DO NOT OVERFILL centrifuge tubes! (*only allowed two) You will need to discard some of the volume. Always note any discarded volumes for correct back calculations. • Record volume of 1S crude extract (supernatant) fraction • Collect in 50 mL conical tubes *Leave some room for freezing! • Take 1 mL 1S aliquot to use for activity & dye‐binding assays Label all tubes before turning in to TFs! Always keep samples & aliquots on ice! Part B: NAD+ & NADH spectra Steps: 1) Obtain spectra from 250 to 400 nm for NAD+ & solutions on Cary‐60 UV/Vis stations *Use UV plastic cuvettes 2) Record NAD+ and absorbance readings on benchtop specs at 340 nm *Use glass test tubes and the same samples you used from the Cary‐60 Use the dilution scheme in Chapter 3 part B Cofactors of LDH ● LDH uses pyridine nucleotide coenzymes as cofactors to transfer reducing equivalents ● ● H+ and a pair of electrons NAD+ – Nicotinamide adenine dinucleotide  Oxidized form  Reduced form ● Pyridine nucleotides have characteristic absorbance spectra ● NAD+: No absorbance at 340 nm : λmax = 340 nm, ε = 6210 M‐1cm‐1 Purpose of NAD+ & NADH spectra ● To calculate εapp for benchtop spectrophotometer since using these specs to measure enzyme activity – – • Given ε340 for NADH (6210 M‐1 cm‐1) is for rectangular cuvettes with 1 cm path length When test tube is used, light path length (l) may differ  Recall Beer’s Law! A = ε l c Measure A340 of NADH on both benchtop spec & UV‐Vis spec, then calculate: ε340(apparent) = [ε340(UV)] x [A340(test tube)/A340(UV cuvette)] How to Measure Activity of LDH ● ● Enzymes are catalysts – speed up the rate of the reactions Activity assay measures: ● Rate of consumption of reactant (NADH) ● NADH has a visible absorbance at 340 nm – can follow its rate of oxidation as reaction progresses  ΔA340/min