Lecture 3 Flashcards
What are hydrogen bonds?
Electrostatic attractions between a H-bond donor and an acceptor.
What is a donor?
Highly polar -OH or -NH groups are good H-bond donors (a H-atom that is covalently bonded to another EN atom)
What is an acceptor?
An electronegative atom with available lone pairs of electrons, such as O or N
How strong is the hydrogen bond to a covalent bond?
About 5-10% strong, enough to make molecule R1 stick to R2 but not form a permanent link
What are H-bonds?
Directional - stronger if donor and acceptor line up with one another.
What are the amino acids with positive side chains?
His, Lys, Arg
What are the characteristics of positively charged side chains? d
- The side chains contain weak bases that gain H+ (become protonated) and so are positively charged in aqueous solution at neutral pH
- The charge makes them very polar, overriding the non-polar hydrocarbon side chain.
What amino acids have negatively charged side chains?
Asp and Glu
What are the characteristics of the negatively charged side chains?
They have carboxylic acid groups R-COOH that lose H+ (become protonated) at neutral pH
What is COOH deprotonated?
Described as carboxylate groups R-COO-
What are carboxylate groups?
Negative and very polar
What is the Asp side chain?
-CH2-COO-
What is the Glu side chain?
-CH2-CH2-COO-
What can amino acids act as?
Acids and Bases
What does an acid do?
Donote a proton
What does a base do?
Gain a proton
What will the acids and bases do?
Gain (protonate) or lose (deprotonate) H+ depending on availability of H+ in solution.
What do natural biochemical occur close to?
pH 7
What is the equation for how pH express availability of H+:
pH = -log10[H+]
at pH = 7, [H+] = 10-7 M
What does the Henderson-Hasselbalch equation relate?
pH, pKa and the state of ionization of a given group.
What is the Henderson-Hasselbalch equation?
pH = pKa + log[deprotonated]/[protonated]
What do the vast majority of molecules for the a-carboxylate group at pH 7 exist as?
As carboxylate -COO NOT as carboxylic acid - COOH (in deprotonated form)
What do the vast majority of molecules for the a-amino group at pH 7 exist as?
As protonated RNH3+ rather than as RNH2
What is the correct structure to represent an individual amino acid at neutral pH?
A dipolar ion (zwitterion)
What happens when the amino acid is part of a peptide chain?
The a-amino groups and the a-carboxylate groups are linked as uncharged amide (except for the ones at the N and C termini, which are also NH3+ and COO- at pH 7)
Which amino acids have ionizable side chains?
There are SEVEN amino acids with side chains that have acid/base properties.
What are the amino acids that have acid/base properties for side chains?
Aspartate, Glutamate, Tyrosine, Cysteine, Arginine, Histidine, Lysine
What are the side chains of Arg, His and Lys?
Neutral when deprotanted and positive when protonated
What does the value of pKa tell you?
Where in the pH scale a group undergoes deprotonation
What can a molecule have?
Several ionizable groups, that has its own Pka value dependent on its chemical context.
When an amino acid is part of a peptide chain, what will happen?
It will have a slightly different pKa
What happens if the pH is one unit or more below the pKa:
The group is fully protonated
What happens if the pH is one unit or more higher than pKa?
The group is fully deprotanated
What happens if the pH is equal to pKa?
The group is 50% deprotonated and 50% protonated
What happens if the pH is less then one unit away from pKa?
Use the Henderson-Hasselbalch equation
What can amino acid analysis determine?
Help to determine protein structure
What does analysis of amino acids involve?
1.) Separation of a mixture into components
2.) Detection of the components of interest
- Can be qualitative (tells you what’s present)
- Can be quantitivate (tells you how much is present)
- Can be preparative (separated components can be recovered for further experiments)
Particles of a solid are chosen with a specific property (eg, silica gel)
Known as the stationary phase - polar.
Liquid solvent or buffer flows past the particles and is non-polar
Mobile phase
Amino acids exchange (partition) between phases
- Polar amino acids P spend more of their time hydrogen bonded to silica and move slowly
- Non-polar amino acids N spend more time in solvent, and move almost as fast as solvent
What is the highest point reached by solvent?
Solvent Front
What do very polar amino acids have?
Low Rf
What do non-polar amino acids have?
High Rf - Run with the gel
How can amino acids be detected?
Adding ninhydrin which reacts with secondary and primary amines.
Alternate is adding fluroscamine, giving yellow fluroscence under UV light.
What does Ion exchange chromatography do?
Separates on the basis of charge
- Uses charged resins as stationary phase
What do cation exchange resins contain?
Negative groups, which bind positive molecules (cations) - Cation Exchange
What do anion exchange resins contain?
Negative molecules, which bind negative molecules (anions) - Anion Exchange
What is Elution by?
- Competition with a high ion concentration (usually NaCl), which displaces the amino acid from the resin
- Changing the pH to alter the charge on the amino acid, so it no longer binds to the resin.
