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Questions and Answers
What is the primary principle behind the separation of amino acids in this technique?
What is the primary principle behind the separation of amino acids in this technique?
- Differences in temperature resistance of amino acids
- Differences in chemical structure of amino acids
- Differences in molecular weight of the amino acids
- Differences in charge and its magnitude of the amino acids (correct)
At pH 3, what state do most amino acids exist in during the separation process?
At pH 3, what state do most amino acids exist in during the separation process?
- Cationic state (correct)
- Anionic state
- Zwitterionic state
- Neutral state
How do positively charged amino acids interact with the negatively charged resin in the column?
How do positively charged amino acids interact with the negatively charged resin in the column?
- They are repelled by the resin and pass through quickly
- They dissolve into the resin matrix
- They are chemically transformed by the resin
- They are attracted to the resin and adsorbed onto it (correct)
Which amino acids will have more affinity for the negatively charged resin particles?
Which amino acids will have more affinity for the negatively charged resin particles?
What will happen to amino acids with least positive charges at pH 3 during separation?
What will happen to amino acids with least positive charges at pH 3 during separation?
What happens to proteins that have a net positive charge in an electric field?
What happens to proteins that have a net positive charge in an electric field?
Which factor does NOT affect the velocity of migration of proteins in an electric field?
Which factor does NOT affect the velocity of migration of proteins in an electric field?
At what point does electrophoretic mobility of a protein become zero?
At what point does electrophoretic mobility of a protein become zero?
How do molecules of the same charge but different molecular masses behave in an electric field?
How do molecules of the same charge but different molecular masses behave in an electric field?
Which component is essential for the conducting liquid in SDS-PAGE?
Which component is essential for the conducting liquid in SDS-PAGE?
What is the role of frictional resistance in the migration of proteins?
What is the role of frictional resistance in the migration of proteins?
How do polar organic molecules behave in paper chromatography compared to non-polar solvents?
How do polar organic molecules behave in paper chromatography compared to non-polar solvents?
What type of substances are used as support in SDS-PAGE?
What type of substances are used as support in SDS-PAGE?
What procedure can be used to identify points of overlap in a polypeptide chain?
What procedure can be used to identify points of overlap in a polypeptide chain?
What byproduct is produced during the fermentation of lactose by bacteria in milk?
What byproduct is produced during the fermentation of lactose by bacteria in milk?
What happens to the pH of milk as lactic acid concentration increases?
What happens to the pH of milk as lactic acid concentration increases?
Which chemical agent hydrolyzes peptide bonds more effectively in certain amino acids pairs?
Which chemical agent hydrolyzes peptide bonds more effectively in certain amino acids pairs?
What type of amino acids does trypsin specifically cleave in peptide bonds?
What type of amino acids does trypsin specifically cleave in peptide bonds?
What is the consequence of blood proteins becoming isoelectric in an acidic environment?
What is the consequence of blood proteins becoming isoelectric in an acidic environment?
Which method does cyanogen bromide (CNBr) employ to cleave peptide bonds?
Which method does cyanogen bromide (CNBr) employ to cleave peptide bonds?
How does extreme pH affect protein structure?
How does extreme pH affect protein structure?
What effect does urea have on proteins?
What effect does urea have on proteins?
Which of the following peptide bonds does chymotrypsin specifically target?
Which of the following peptide bonds does chymotrypsin specifically target?
Which of the following amino acids does thermolysin cleave in addition to those cleaved by chymotrypsin?
Which of the following amino acids does thermolysin cleave in addition to those cleaved by chymotrypsin?
What is the role of mercaptoethanol in protein denaturation?
What is the role of mercaptoethanol in protein denaturation?
What happens to methionine after hydrolysis using cyanogen bromide?
What happens to methionine after hydrolysis using cyanogen bromide?
Which type of pH condition results in greater unfolding of proteins?
Which type of pH condition results in greater unfolding of proteins?
Which method is commonly used in laboratories to cleave peptide bonds besides chemical methods?
Which method is commonly used in laboratories to cleave peptide bonds besides chemical methods?
What is a common result of protein denaturation in biological systems?
What is a common result of protein denaturation in biological systems?
What happens to proteins at their isoelectric point (pI)?
What happens to proteins at their isoelectric point (pI)?
How can different proteins be separated based on their solubility characteristics?
How can different proteins be separated based on their solubility characteristics?
What effect does pH have on the solubility of β-lactoglobulin?
What effect does pH have on the solubility of β-lactoglobulin?
Which of the following factors does NOT influence protein solubility?
Which of the following factors does NOT influence protein solubility?
What is the primary reason for the increased solubility of proteins outside their pI?
What is the primary reason for the increased solubility of proteins outside their pI?
What is the characteristic of a protein's solubility curve?
What is the characteristic of a protein's solubility curve?
Which mechanism describes the separation of bound proteins using substrates?
Which mechanism describes the separation of bound proteins using substrates?
Why do proteins precipitate at their isoelectric point, even with positive and negative charges present?
Why do proteins precipitate at their isoelectric point, even with positive and negative charges present?
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Study Notes
Milk Fermentation and Coagulation
- Bacteria grow in stored milk, using lactose for energy during fermentation.
- Lactic acid is produced, decreasing the milk's pH.
- Lower pH causes milk proteins to become isoelectric and coagulate, forming curd.
Blood Protein Denaturation
- Acidic or basic blood pH leads to denaturation of proteins like albumin and fibrinogen.
- Denatured proteins lose functionality, risking oxygen transport by hemoglobin and survival.
Protein Unfolding at Extreme pH
- High net charge at extreme pH causes swelling and unfolding of proteins.
- Extreme alkaline conditions ionize buried groups, disrupting polypeptide structures more than extreme acidic conditions.
Organic Solvent Effects on Proteins
- Detergents (e.g., sodium dodecyl sulfate) and organic solvents (e.g., alcohol, acetone) denature proteins.
- Urea forms stronger hydrogen bonds with proteins, disrupting internal bonding.
- Mercaptoethanol reduces disulfide bonds, altering three-dimensional protein structures.
Separation of Amino Acids
- Separation is based on charge differences (positive, negative, neutral) of amino acids.
- At pH 3, amino acids behave predominantly as cations and are exchanged for sodium ions in cation resin columns.
- Basic amino acids have a stronger affinity for negatively charged resin, affecting separation rates.
Hydrolysis Techniques
- Acidic hydrolysis can cleave peptide bonds but shows varying susceptibility among amino acids.
- Cyanogen bromide specifically cleaves methionine-containing bonds, transforming methionine to homoserine lactone.
- Proteolytic enzymes like trypsin and chymotrypsin hydrolyze bonds based on specific amino acid characteristics.
Protein Separation Methods
- SDS-PAGE uses a strong electric field to separate proteins based on charge and size.
- Proteins migrate towards opposite electrodes; net charge determines direction and speed.
- Velocity of migration depends on electric field strength, protein charge, and frictional resistance.
Isoelectric Point and Separation
- At isoelectric pH, protein charge is neutral, resulting in zero electrophoretic mobility.
- Different proteins will migrate at varying rates based on charge and size.
- Polarity influences chromatography; polar molecules prefer aqueous environments over non-polar solvents.
Solubility and Salting-Out of Proteins
- Protein solubility varies with pH; proteins are least soluble at their isoelectric points (pI).
- Salting-out techniques further exploit solubility differences influenced by pH and ionic strength.
- β-lactoglobulin, a milk protein, has a pI of 5.3; it's soluble at extreme pH levels and precipitates at its pI.
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