Enzyme Kinetics & Catalysis
What is the role of NADH in the LDH reaction, and how is it monitored during assays?
NADH acts as an electron donor in the LDH-catalyzed conversion of pyruvate to lactate. It is monitored by measuring its decrease in absorbance at 340 nm.
Why is ammonium sulfate added slowly and with stirring during protein precipitation?
To avoid local high salt concentrations that can denature proteins and to ensure uniform precipitation.
What equation describes the relationship between absorbance, concentration, and path length in spectrophotometry?
Beer-Lambert Law: A = ε × c × l (Absorbance = molar extinction coefficient × concentration × path length).
Why is balancing centrifuge tubes essential before starting a centrifugation run?
To prevent rotor imbalance that can damage the centrifuge or cause tube breakage.
Why is it important to aliquot protein samples before freezing for storage?
To avoid repeated freeze-thaw cycles, which can denature proteins and reduce activity.
What does Km represent in enzyme kinetics, and what does a low Km indicate about enzyme-substrate affinity?
Km is the substrate concentration at which the reaction rate is half of Vmax. A low Km indicates high enzyme-substrate affinity.
What is the function of EDTA in homogenization buffers when preparing tissue extracts?
EDTA chelates divalent metal ions, inhibiting metalloproteases and preventing degradation of the protein of interest.
What is the recommended absorbance range for reliable measurements using the Spec 20 spectrophotometer?
Between 0.1 and 1.0 absorbance units.
What could happen to your enzyme if you over-homogenize or overheat your tissue extract during blending?
Proteins can denature due to heat buildup or mechanical shear, leading to loss of activity.
What controls should you include in an LDH enzyme activity assay to ensure valid results?
A blank with no enzyme, a positive control with known enzyme activity, and possibly a negative control without substrate.
What happens to the rate of an enzyme-catalyzed reaction when the substrate concentration is much greater than Km?
The enzyme is saturated, and the reaction proceeds at Vmax—further increases in substrate concentration won’t significantly increase the rate.
Why is it critical to keep protein samples on ice during purification and extraction steps?
To minimize protease activity and prevent heat-induced protein denaturation.
Why do we use a blank when taking absorbance readings in spectrophotometry, and what should it contain?
To zero the instrument and correct for background absorbance; it should contain everything except the analyte (e.g., buffer and reagents minus the protein or NADH).
You run an LDH assay and see no decrease in absorbance at 340 nm, what are three possible reasons?
Substrate or coenzyme (NADH) degradation, incorrect assay setup (e.g., missing enzyme), or enzyme denaturation/inactivity.
How does pH affect protein purification and enzyme activity, and why must buffer pH be carefully chosen?
pH affects protein solubility and enzyme active site protonation state; incorrect pH can lead to precipitation or loss of activity.
In competitive inhibition, how are Km and Vmax affected, and why?
Km increases because the inhibitor competes with the substrate for the active site; Vmax remains unchanged because high substrate concentrations can overcome the inhibition.
After ammonium sulfate precipitation, why do we perform dialysis on protein samples before further purification?
To remove excess salts (like ammonium sulfate) that can interfere with downstream purification steps or assays.
Why is 340 nm the optimal wavelength to measure NADH consumption in an LDH assay?
NADH has a strong absorbance peak at 340 nm, while NAD+ does not, making it ideal to track NADH oxidation.
Your SDS-PAGE gel shows smeared protein bands—what might have gone wrong in your sample preparation?
Overloading protein, incomplete denaturation (insufficient heating or reducing agent), or degradation of the protein sample.
What steps would you take to confirm whether your LDH sample contains both M-type and H-type isoenzymes?
Run an isoenzyme separation via electrophoresis or chromatography and compare activity profiles under different substrate/cofactor conditions.
How can you distinguish between competitive and noncompetitive inhibitors using a Lineweaver-Burk plot?
Competitive inhibition shows lines intersecting on the y-axis (Vmax unchanged), while noncompetitive inhibition shows lines intersecting on the x-axis (Km unchanged, Vmax decreases).
What three steps would you include in a purification protocol for isolating an intracellular enzyme from tissue?
Homogenization with buffer (cold, with EDTA), ammonium sulfate precipitation (two-step cut), dialysis followed by affinity or ion-exchange chromatography.
What factors could cause an unreliable standard curve in a Bradford assay, and how can you address them?
Protein precipitation, improper mixing, expired reagents; fix by ensuring fresh standards, proper pipetting, and mixing immediately before reading absorbance.
After dialysis, your protein precipitates out of solution—what are two possible causes and how can you prevent this?
Rapid removal of stabilizing salts or wrong pH in dialysis buffer; prevent by optimizing dialysis buffer conditions, adding glycerol, or lowering temperature.
What are three strategies to troubleshoot low activity after purifying an enzyme by affinity chromatography?
Check for proteolysis (use inhibitors), ensure proper elution conditions (pH, salt), and verify the enzyme wasn’t denatured by elution conditions (e.g., harsh buffers).