30 Questions
What happens to glutamate side chain at a lower pH compared to a higher pH?
Remains protonated at lower pH, deprotonates at higher pH
What happens to arginine side chain at a higher pH compared to a lower pH?
Deprotonates at higher pH, remains protonated at lower pH
Which type of group is present in the side chain of arginine amino acid?
Guanidinium group
In basic conditions, what happens to negatively charged side chains?
They become neutral
At what pH does the carboxylic acid group in glutamate start to deprotonate?
Above its pKa
What is the role of hydrogen ions in the side chains' ionization process?
Facilitate protonation
Which aromatic amino acid has the strongest absorbance peak around 280 nm?
Tryptophan
What does the height of the peak or the intensity of absorbance correlate with in a protein sample?
The concentration of proteins in the sample
Which of the following amino acids is not typically found in the interior of proteins, away from water-exposed surfaces?
Serine
What is the significance of the specific wavelengths at which proteins absorb UV light most strongly?
They depend on the specific aromatic amino acids present in the protein sequence
What technique is used to analyze the concentration and purity of proteins in a sample?
UV Absorbance
Which of the following amino acids has side chains containing alcohol (-OH) groups?
Serine
Which amino acid contains a pyrroline ring as part of its side chain?
Pyrrolysine
Which amino acid derivative is used as the initial amino acid in the synthesis of proteins in prokaryotes and in mitochondria of eukaryotes?
N-Formylmethionine
Which amino acid analogue has a selenium atom (-SeH) replacing the sulfur atom in cysteine?
Selenocysteine
What is the approximate pKa value of the amino group (NH2) in amino acids?
9
What is the pKa value of the carboxyl group (COOH) in amino acids?
2
At physiological pH (around 7.4), most amino acids have a net charge close to which of the following?
0
What is the formula for calculating the isoelectric point (pI) of an amino acid?
pI = (pKa of carboxyl group + pKa of amino group) / 2
Which amino acids may have a third pKa value associated with their side chain ionization in pI calculations?
Histidine, lysine, and arginine
In the Henderson-Hasselbalch equation, what does [A-] represent?
Concentration of the conjugate base
What mainly contributes to the charges in a polypeptide chain after polymerization?
Side chains of amino acids
'Chains of linked amino acids' is known as what in the context of polypeptides?
Proteins
How are individual residues in a polypeptide chain identified?
By their 1-letter or 3-letter codes
What is the main property that Hydrophobic Interaction Chromatography (HIC) utilizes for molecule separation?
Hydrophobicity
In Size Exclusion Chromatography (SEC), which molecules elute first?
Large molecules
What is a major advantage of Affinity Chromatography over other chromatography techniques?
It operates under physiological conditions
What type of molecules get trapped in the porous beads in Size Exclusion Chromatography?
Small molecules
Which chromatography technique uses gradient elution to change the composition of the mobile phase over time?
Hydrophobic Interaction Chromatography (HIC)
In Affinity Chromatography, what is the binding material that interacts with the desired protein?
Antigen
Study Notes
Amino Acid Properties
- Aromatic amino acids (tryptophan, tyrosine, phenylalanine) absorb UV light at specific wavelengths, allowing for protein concentration and purity analysis
- UV absorbance graphs show peaks at specific wavelengths, correlating with protein concentration
Amino Acid Types
- Alcohol-containing amino acids: serine, threonine, asparagine, glutamine
- Other amino acids:
- Homoserine: derivative of serine
- Homocysteine: derivative of cysteine
- N-Formylmethionine: used in protein synthesis in prokaryotes and eukaryotic mitochondria
- Selenocysteine: analogue of cysteine with selenium instead of sulfur, involved in antioxidant functions
- Pyrrolysine: uncommon amino acid found in certain archaea and bacteria, used in specific proteins
Amino Acid Ionization
- Amino acids undergo ionization due to acidic (carboxyl) and basic (amino) groups, leading to different forms depending on pH
- Amino acids typically have two pKa values, corresponding to the ionization of their acidic and basic groups
- Isoelectric point (pI) is the pH at which an amino acid carries no net charge, calculated as pI = (pK1 + pK2) / 2
Polypeptides
- Chains of linked amino acids, known as residues, with each residue identified by its 3-letter or 1-letter code
- Amino acids in a polypeptide chain lose most of their charges from their carboxyl and amino groups due to polymerization
- Resulting charges mainly come from the side chains of the amino acids
Ionization of Side Chains
- Side chains of amino acids can undergo ionization, transitioning between protonated (acidic) and deprotonated (basic) forms depending on pH
- Positively charged side chains gain a proton in acidic conditions, becoming neutral
- Negatively charged side chains lose a proton in basic conditions, becoming neutral
Specific Amino Acids
- Glutamate: contains a carboxylic acid group in its side chain, which can be protonated or deprotonated depending on pH
- Arginine: contains a guanidinium group in its side chain, which can be protonated or deprotonated depending on pH
Chromatography Techniques
Size Exclusion Chromatography (SEC) / Gel Filtration Chromatography
- Separates proteins based on size and shape
- Utilizes porous beads in the column that allow smaller molecules to enter and take longer paths, while larger molecules pass through more quickly
- Larger molecules elute first, followed by smaller ones
- Gentle on biomolecules and doesn't require a mobile phase
Affinity Chromatography
- Antibody-antigen interaction: desired protein binds to specific antigen in the column, while non-binding proteins pass through
Test your knowledge on proteins and UV absorbance with this quiz. Learn about the role of aromatic amino acids like tryptophan, tyrosine, and phenylalanine in absorbing UV light, and how this technique is used to analyze protein concentration and purity.
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