Protein Purification, Enzyme Assays and Inhibition

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Questions and Answers

Describe in two sentences the main principle behind affinity chromatography and give a specific example of a ligand-enzyme pair used in this technique.

Affinity chromatography separates proteins based on their specific binding affinity to an immobilized ligand. An example is using immobilized Ni2+ ions to purify His-tagged proteins.

Explain in two sentences how a coupled enzyme assay works and why it is necessary for measuring the activity of certain enzymes. Provide an example.

A coupled enzyme assay links the reaction of interest to a secondary reaction that produces a readily detectable product. This is necessary when the primary reaction's product is difficult to measure directly, like in the hexokinase reaction coupled to glucose-6-phosphate dehydrogenase.

Differentiate between competitive and non-competitive enzyme inhibition in terms of their effects on the Michaelis-Menten constant ($K_m$) and the maximum reaction velocity ($V_{max}$).

Competitive inhibition increases $K_m$ while $V_{max}$ remains unchanged, as the inhibitor competes with the substrate for binding to the active site. Non-competitive inhibition decreases $V_{max}$ while $K_m$ remains unchanged, as the inhibitor binds elsewhere and reduces the enzyme's catalytic efficiency.

Describe how two-dimensional gel electrophoresis (2-D PAGE) separates proteins. What are the two separation principles used in 2-D PAGE?

2-D PAGE separates proteins based on their isoelectric point (pI) in the first dimension using isoelectric focusing (IEF), followed by separation based on size in the second dimension using SDS-PAGE. Signup and view all the answers

Explain in 2-3 sentences the basic principle of size exclusion chromatography (SEC) and its primary use in protein purification or characterization.

Size exclusion chromatography separates molecules based on their size. The stationary phase consists of porous beads. Smaller molecules enter the pores later and elute later, while larger molecules cannot enter the pores and elute earlier. It is primarily used to estimate the molecular weight of proteins or to separate proteins from smaller contaminants. Signup and view all the answers

When purifying intracellular proteins, what is the purpose of cell disruption, and what are two key considerations for optimizing this process?

The purpose of cell disruption is to break open cells to release intracellular proteins. Two key considerations are extraction buffer preparation and protease inhibitors. Signup and view all the answers

What are inclusion bodies, and what is a major challenge associated with purifying proteins from them?

Inclusion bodies are insoluble aggregates of overexpressed proteins found in cells. A major challenge is protein renaturation after isolation. Signup and view all the answers

Why is it important to consider protease inhibitors during cell disruption, and what problem do they prevent?

Protease inhibitors are important because they prevent degradation of the target protein by proteases released during cell lysis. Signup and view all the answers

Describe the role of reducing agents like DTT in protein purification, and explain the chemical principle behind their use.

Reducing agents like DTT maintain proteins in a reduced state by donating electrons, preventing the formation of disulfide bonds. The principle is to keep cysteine residues from oxidizing and forming unwanted bonds. Signup and view all the answers

What challenges are typically encountered during the purification of extracellular proteins, and how do those challenges differ when working with intracellular proteins?

Extracellular proteins are typically present in low amounts and in dilute solutions that can be difficult to concentrate, while intracellular proteins may be present in large amounts but mixed with lipids and proteases. Signup and view all the answers

Explain how combining multiple protein purification techniques can sometimes lead to a loss of the target protein, despite their individual effectiveness.

Each purification step, while effective on its own, can introduce losses due to handling, non-specific binding, or incomplete recovery. Combining multiple steps multiplies these potential losses, reducing the overall yield of the target protein. Signup and view all the answers

Describe a scenario where pre-cleaning a protein extract by precipitation might be more advantageous than direct chromatography, considering potential downstream effects.

Pre-cleaning by precipitation can remove a large portion of unwanted proteins and contaminants early on, which protects chromatography columns from fouling and increases their lifespan and capacity for the target protein. Signup and view all the answers

A protein sample has a total activity of 10,000 U and a total protein content of 50 mg after the initial cell lysis step. After affinity chromatography, the total activity is 8,000 U and the total protein content is 8 mg. Calculate the specific activity at both stages.

Initial specific activity: 200 U/mg. Specific activity after affinity chromatography: 1000 U/mg. Signup and view all the answers

Explain why it's important to empirically determine the efficacy of each step in a protein purification protocol, rather than relying solely on theoretical expectations.

Theoretical expectations may not account for protein-specific behaviors or variations in sample composition. Empirical determination allows for identifying steps that aren't performing as expected, enabling optimization or replacement with more effective techniques. Signup and view all the answers

Describe how the choice of starting material (tissue or cell type) can influence the subsequent steps in a protein purification strategy.

Different tissues or cells may contain varying levels of the target protein and different sets of contaminating proteins. This will require adjustments to lysis, pre-cleaning, and chromatography steps to effectively isolate the protein of interest. Signup and view all the answers

Explain how knowledge of a protein's characteristics (e.g., post-translational modifications) helps to optimize purification.

Knowledge of post-translational modifications, such as glycosylation or phosphorylation, can enable the use of specific affinity chromatography methods that target these modifications, leading to more efficient purification. Signup and view all the answers

A researcher is purifying a recombinant protein and observes significant proteolytic degradation during the cell lysis step. Describe two specific strategies they could implement to minimize this degradation.

<ol> <li>Add a cocktail of protease inhibitors to the lysis buffer to block proteolytic activity.</li> <li>Perform the lysis step at a low temperature (e.g., 4°C) to slow down enzymatic reactions.</li> </ol> Signup and view all the answers

A protein has a high affinity for divalent metal ions. Outline a purification strategy that exploits this property and explain why it would be effective.

Use Immobilized Metal Affinity Chromatography (IMAC). IMAC involves a resin with immobilized metal ions (e.g., Ni2+). The protein will bind to the column due to its affinity for the metal ions, while other proteins will wash through. The bound protein can then be eluted using a buffer containing a competing metal chelator (e.g., imidazole). Signup and view all the answers

In discontinuous electrophoresis, what is the primary purpose of the stacking gel in relation to protein separation?

The stacking gel concentrates the proteins into narrow bands before they enter the resolving gel, leading to improved resolution. Signup and view all the answers

How does SDS contribute to the separation of proteins during SDS-PAGE electrophoresis?

SDS denatures proteins and coats them with a negative charge, ensuring that separation is based on size rather than intrinsic charge. Signup and view all the answers

Explain the relationship between the logarithm of a protein's molecular weight (log Mr) and its migration rate in SDS-PAGE. What kind of relationship is observed?

There is a linear inverse relationship between the log of a protein's molecular weight and its relative migration in the gel. Signup and view all the answers

In isoelectric focusing (IEF), how is a stable pH gradient established and maintained within the gel?

A stable pH gradient is established using ampholytes or Immobilines, which are incorporated into the polyacrylamide matrix. Signup and view all the answers

Describe the two separation principles that are combined in two-dimensional electrophoresis (2D-PAGE).

2D-PAGE combines isoelectric focusing (IEF), separating by isoelectric point, and SDS-PAGE, separating by molecular weight. Signup and view all the answers

In capillary electrophoresis (CE), what replaces the gel matrix used in traditional electrophoresis techniques?

In CE, a liquid electrolyte solution within a narrow capillary tube replaces the traditional gel matrix. Signup and view all the answers

Define electroendosmotic flow (EOF) and briefly describe its role in capillary electrophoresis (CE).

EOF is the movement of liquid through a capillary due to the interaction of the charged capillary wall and the buffer solution. It helps to move all solutes, regardless of their charge, towards one electrode. Signup and view all the answers

Outline the purpose of a Western blot and explain how it builds upon the results of gel electrophoresis.

A Western blot is used to detect specific proteins from a sample after they have been separated by gel electrophoresis, allowing for identification and quantification of the target protein. Signup and view all the answers

How does the Bradford assay leverage the properties of proteins to enable quantification, and what limitations arise from this method?

The Bradford assay uses the binding of Coomassie Brilliant Blue G-250 to basic and aromatic amino acids to measure protein concentration. Aromatic amino acids include Arg, Lys, His, Phe, Tyr, Trp. The subjectivity and size limitation (3-5 kDa) impacts accuracy. Signup and view all the answers

Explain the fundamental principle behind the Biuret assay for protein quantification, and outline its major advantage and disadvantage.

The Biuret assay depends on a color change reaction with peptide bonds and copper sulfate. Advantage: It is objective. Disadvantage: It has low sensitivity. Signup and view all the answers

Why is differential measurement (Whitaker and Granum method) better than direct measurement at $280\ nm$?

Differential measurements at $235\ nm$ and $280\ nm$ are better because they account for proteins with fewer aromatic rings. Aromatic rings have a $\pi-\pi*$ absorption, which you can use to quantify the amount of protein in a sample. Signup and view all the answers

Describe how the BCA assay combines the principles of the Biuret assay, and what benefits does this combination provide?

The BCA assay combines the Biuret reaction with bicinchoninic acid (BCA) to increase sensitivity. The Biuret reaction relies on reducing amino acids: Tyr, Trp, Cys. This makes it more tolerant to interfering substances. The BCA assay also works in the presence of detergents, and can be used on a micro scale. Signup and view all the answers

In fluorescence assays with OPA, what specific amino acid residues primarily react with OPA, and under what pH conditions is this reaction optimal?

OPA reacts primarily with Lys and Arg residues at alkaline pH. Signup and view all the answers

For a protein containing a metal center, describe how its concentration can be determined without relying on amino acid composition.

Proteins with metal centers or chromophores can be quantified by measuring light absorbance. Chromophores absorb visible light. Signup and view all the answers

Compare and contrast the advantages of using UV absorbance at 205 nm versus 280 nm for protein quantification. What are the underlying principles and limitations of each?

205nm absorption is due to the peptide bond (objective). 280nm absorption is due to aromatic amino acids (less objective). 205nm has more interfering substances and 280nm is affected by aromatic amino acid composition. Signup and view all the answers

When selecting a method for protein quantification, what considerations should be taken into account regarding potential interfering substances in the sample?

Assays have different tolerances for interfering buffer compounds. For example, the BCA assay has a very high tolerance. It is important to consider the other substances in the sample. Signup and view all the answers

A researcher needs to quantify a very dilute protein sample with potential reducing agents. Which assay (Biuret, BCA, or Bradford) would be the most appropriate choice, and why?

The BCA assay would be the most appropriate choice because it is very sensitive and works in the presence of detergents. The Bradford assay is not appropriate because it cannot be used with reducing agents. Signup and view all the answers

Before beginning protein purification from a cellular source, what key preliminary questions should one address to optimize the purification strategy?

What is the starting material, cells or tissue? Is the protein related to primary or secondary metabolism? Signup and view all the answers

Explain how the resolution (R) in liquid chromatography is affected by both the selectivity and the efficiency of the separation process.

Resolution (R) is directly proportional to both selectivity and the square root of efficiency. Improved selectivity (ability to distinguish between compounds) and higher efficiency (narrower peaks) both lead to better resolution, allowing for better separation of two peaks. Signup and view all the answers

Describe how hydrophobic interaction chromatography (HIC) separates proteins based on their hydrophobic properties. Explain the conditions used for binding and elution.

HIC separates proteins based on their surface hydrophobicity. Proteins bind to the HIC resin under high salt concentrations, which enhances hydrophobic interactions. Elution is achieved by decreasing the salt concentration, weakening the hydrophobic interactions and releasing the proteins. Signup and view all the answers

In size exclusion chromatography (SEC), also known as gel filtration chromatography, how are molecules separated, and what property of the molecules determines their elution order?

In SEC, molecules are separated based on their size. Larger molecules elute earlier because they cannot enter the pores in the stationary phase and thus traverse a shorter path. Smaller molecules enter the pores, increasing their path length and eluting later. Signup and view all the answers

Explain the principle behind affinity chromatography and describe the key components involved in this technique.

Affinity chromatography separates proteins based on a specific, reversible interaction between a target protein and an immobilized ligand. Key components include the ligand, the matrix to which the ligand is attached, and the target protein. Signup and view all the answers

Describe the purpose of using a His6 tag in immobilized metal ion affinity chromatography (IMAC) and outline the steps involved in protein binding and elution.

The His6 tag is a sequence of six histidine residues added to a recombinant protein to facilitate purification by IMAC. The His6 tag binds to metal ions (like nickel) immobilized on the resin. Elution is typically achieved by adding imidazole, which competes with the His6 tag for binding to the metal ions, releasing the tagged protein. Signup and view all the answers

What is the role of the carrier matrices in electrophoresis, and what are the two main types of matrices used?

Carrier matrices in electrophoresis provide a medium for separating molecules based on size and charge while preventing convective mixing. The two main types of matrices are agarose and polyacrylamide. Signup and view all the answers

How do the properties of agarose gels make them suitable for separating large molecules like DNA?

Agarose gels have a relatively large pore size that can be adjusted by changing the agarose concentration. This, along with its property as a linear polymer, makes agarose suitable for separating large molecules such as DNA. Signup and view all the answers

Describe the function of the cross-linker N, N-methylene bisacrylamide (Bis) in polyacrylamide gel electrophoresis.

Bisacrylamide acts as a cross-linker to form pores within the polyacrylamide gel. The concentration of Bis affects the pore size and thus the separation properties of the gel. Signup and view all the answers

Explain the relationship between electrophoretic mobility, charge, viscosity, and Stokes radius according to the equation: $u = q / (6\pi\eta r)$

Electrophoretic mobility ($u$) is directly proportional to the charge ($q$) of the molecule and inversely proportional to the viscosity ($\eta$) of the medium and the Stokes radius ($r$) of the molecule. Higher charge increases mobility, while greater viscosity or size decreases mobility. Signup and view all the answers

In ion exchange chromatography, how would you elute a negatively charged protein bound to an anion exchange column?

To elute a negatively charged protein bound to an anion exchange column, you can increase the concentration of a competing anion (e.g., chloride ions) in the buffer, or increase the ionic strength of the buffer. The increased concentration of anions will displace the bound protein, leading to elution. Signup and view all the answers

You are performing HIC and observe that your protein of interest does not bind to the column. What adjustments could you make to the binding conditions to promote protein binding?

To promote protein binding in HIC, increase the salt concentration of the binding buffer. Adding salts like ammonium sulfate will enhance hydrophobic interactions between the protein and the resin. Signup and view all the answers

Describe a scenario where size exclusion chromatography (SEC) would be preferred over other chromatography techniques like ion exchange or affinity chromatography.

SEC is preferred when separating proteins based solely on their size, such as for determining oligomeric state or for desalting a protein sample. It is useful when specific charge or affinity interactions are not relevant or when the protein lacks suitable binding partners for affinity purification. Signup and view all the answers

In affinity chromatography, a strong interaction between the target protein and the ligand can sometimes lead to denaturation of the target protein during elution. Suggest a method to elute the protein while minimizing the risk of denaturation.

To minimize denaturation, use a gentle elution method such as competitive elution with a low concentration of free ligand or a gradual change in pH or ionic strength, rather than harsh conditions that could disrupt the protein's structure. Signup and view all the answers

Explain why slow flow rates and long, thin columns promote interaction in the context of affinity chromatography, as suggested by the equation $Ka = [LT] / ([L]*[T])$.

Slow flow rates and long, thin columns increase the residence time of the target protein (T) in the column, allowing more time for interaction with the immobilized ligand (L) to form the complex (LT). This promotes binding and increases the chances of achieving equilibrium, maximizing the interaction and improving the efficiency of the affinity purification. Signup and view all the answers

What is the purpose of staining a polyacrylamide gel after electrophoresis, and name two common staining methods used for visualizing proteins.

Staining is used to visualize the separated proteins in a polyacrylamide gel. Two common staining methods are Coomassie Brilliant Blue staining and silver staining. Signup and view all the answers

Flashcards

Enzyme purification

The process of isolating enzymes from a mixture for study or use.

Liquid chromatography

A technique used to separate components in a liquid mixture based on their interactions with a stationary phase.