Solubilization of HRP Isoenzymes Study

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How does the hydrophobic environment near the haem group of the HRP basic isoenzyme affect its interaction with surfactants?

It inhibits complex formation with the surfactant

It has no effect on complex formation with the surfactant

It favors complex formation with the surfactant, especially at higher protein concentrations (correct)

It requires the addition of citrate to facilitate the forward transfer process

What is the primary function of the surfactant shell layer in reverse micelles?

To increase the citrate concentration and facilitate forward transfer

To chelate Ca2+ ions and improve protein purification

To solubilize water and macromolecules within the hydrophilic core

To protect enzymic proteins from denaturation by the organic phase (correct)

What is a potential biotechnological application of reverse micelles?

Solubilization of hydrophilic proteins, nucleic acids, and other entities (correct)

Denaturation of enzymic proteins to reduce their catalytic activity

Chelation of Ca2+ ions to improve protein purification

Increasing citrate concentration to facilitate forward transfer

What is the effect of back extraction on the activity of proteins solubilized in reverse micelles?

The protein recovers near full activity Signup and view all the answers

What is the effect of the organic phase on the catalytic activity of enzymic proteins in reverse micelles?

The surfactant shell layer protects the proteins from denaturation by the organic phase, resulting in little or no damage to their catalytic activity Signup and view all the answers

What is the purpose of using reverse micelles in protein purification?

To concentrate a single protein as one of the first steps in downstream processing Signup and view all the answers

What was the primary purpose of the first stage of the forward transfer process?

To remove contaminating proteins from the crude extract Signup and view all the answers

What effect did adding citrate to the crude extract have on the purification process?

It improved both the purification factor and yield after the second forward transfer Signup and view all the answers

Why was solubilization of contaminant proteins favored over HRP at pH 2.8?

HRP has a very weak positive surface charge at this pH Signup and view all the answers

What was the purpose of adding citrate to the crude extract?

To chelate $Ca^{2+}$ ions and improve HRP solubilization Signup and view all the answers

What conditions were used for the second forward transfer to solubilize HRP?

pH 3.15, 0.15 M NaCl, $V_{aq}/V_{org}$ = 10 Signup and view all the answers

What was the effect of increasing ionic strength on HRP solubilization at pH 2.8?

It decreased HRP solubilization in the reverse micellar phase Signup and view all the answers

What surfactant was used to produce the reverse-micellar phases in the purification of horseradish peroxidase?

AOT dissolved in isooctane Signup and view all the answers

In the purification process, at what specific pH range did HRP solubilization occur?

Narrow pH range Signup and view all the answers

What was the final specific activity of the purified horseradish peroxidase (HRP)?

86 guaiacol U mg -1 Signup and view all the answers

Which electrophoresis technique showed two overlapping bands corresponding to HRP at 43.8 kDa?

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Signup and view all the answers

Which liquid chromatography technique revealed that most of the specific activity of the purified HRP was due to the basic isoenzyme with pI 8.5?

Ion exchange liquid chromatography Signup and view all the answers

What caused high HRP losses during direct reverse-micellar extraction from the crude extract?

Precipitate at the interface Signup and view all the answers