BIOC 2580 Lecture Notes - Thin-Layer Chromatography, Column Chromatography, Deprotonation

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Published on 14 Apr 2013
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Biochemistry F’12 Notes
Molecules we study in biochemistry
Small molecules
sugars, amino acids, nucleotides, carboxylic acid derivatives
act as building blocks for macromolecules
Macromolecules
Proteins chains of amino acids
Polysaccharides chains of simple sugars
Nucleic acids chains of nucleotides
How large is a protein molecule?
Most proteins: 10,000 to 100,000 g/mol
Protein size is expressed in kiloDaltons (kDa)
o 1 Dalton (Da) = 1 g/mol (mass of H atom)
o 1 kDa = 1000 g/mol
Myoglobin is 16.5 kDa small protein
P-glycoprotein is 170 kDa large protein
Proteins are made of amino acids
linear chains of amino acids
linked by peptide bonds (type of amide bond)
Each protein has:
o unique sequence of different amino acids
o a well-defined size and structure
Proteins have diverse functions including:
o catalyzing reactions (enzymes)
o forming complex subcellular structures
Basic amino acid structure
Each amino acid has an amino group and a carboxylate group
Each amino acid has a different side chain R
20 different amino acids are found in proteins
Peptide bonds
Condensation involves removal of H2O from the units
being linked
Hydrolysis regenerates the original carboxylic acid and
amino group
The C=O group of the amide is the point of weakness
allowing H2O attack
Large numbers of amino acids can be linked together to form a
peptide chain
The combination of different side chains R1, R2, R3, etc
gives each protein its unique properties
There are 153 amino acids in myoglobin (16.5 kDa)
Polypeptide a chain with many amino acids, usually a complete protein
o Greek poly = many
Oligopeptide a chain with a few amino acids, usually a fragment
o Greek oligo = a few
Figure 1Basic structure of an amino acid
Figure 2 Condensation reaction to form peptide bond
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Amino acid side chain structure
Carbon atoms of the amino acid core are identified by
Greek Letters
The α-carbon is the central backbone atom
The β-carbon is the first atom of the side chain, the γ-
carbon is the second, etc
Functional groups may be linked to different core atoms:
o α-amino
o ε-amino
Amino acids with very non-polar side chains Ala, Val, Leu, Ile, Met, Phe
The side chains are dominated by hydrocarbon, and consist only of C-C and C-H
bonds
Hydrocarbon is nonpolar and hydrophobic (or water avoiding)
Polar and non-polar properties
Polarity is a consequence of atoms having different electronegativity or
tendency to hold bonding electrons
o O> N > S > C H
Atoms with similar electronegativity share bonding electrons equally, e.g. C-
C, C-H, and are non-polar
Pairs of atoms with different electronegativity distribute bonding electrons
unequally more electronegative atoms such as O or N get greater than
50% share, and this leads to unbalanced charges and polar bonds
Moderately non-polar: Gly, Cys, Pro, Tyr, Trp
Glycine has single H atom as side chain, not enough to be very non-polar
o Hydrophobicity is related to the number of CH, CH2 or CH3 groups
present
Cysteine contains the slightly polar SH group
Proline is unique because the side chain links to α-N as well as to α-C. The
polar N moderates the non-polar hydrocarbon.
Tyrosine has a single polar group that partly offsets the very non-polar benzene ring.
Tryptophan behaves similarly.
Amino acids with polar uncharged side chains: Ser, Thr, Asn, Gln
Serine and threonine have side chains that include the polar hydroxyl group -OH (simple
alcohol)
Asn and Gln both contain the polar amide group
These side chain groups do not gain or lose H+ in aqueous solution, so they are
uncharged
All four side chains act as good hydrogen bond donors or acceptors
Hydrogen bonds are electrostatic attractions between an H-bond donor and an acceptor
Highly polar OH or NH groups are good H-bond donors
An acceptor is an electronegative atom with an available lone pair of electrons, such
as O or N
The hydrogen bond (- -) is about 5-10% as strong as a covalent bond, enough to
make molecule R1 stick loosely to R2 but not to form a permanent link.
Figure 3 Identification of carbon atoms with greek letters
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Positively charged side chains His, Lys and Arg
These side chains contain weak bases that gain H+ (become protonated) and so are positively charged in
aqueous solution at neutral pH
For example, the lysine side chain
Charge makes them very polar, overriding the non-polar hydrocarbon chain
Negatively charged side chains Asp and Glu
Side chains have carboxylic acid groups R-COOH that lose H+ (become deprotonated) at neutral pH
o When deprotonated these are described as carboxylate groups R-COO-
o Carboxylate groups are negative and also very polar
Asp side chain: -CH2-COO-
Glu side chain: -CH2-CH2-COO-
Free amino acids are weak electrolytes due to their amino and carboxylate groups
Normal biochemical processes occur close to pH 7 (physiological pH is 7.0-7.4)
Groups such as carboxylate and amino groups gain or lose H+ depending on availability of H+ in solution
pH expresses availability of H+:
The Henderson-Hasselbalch equation relates pH, pKa and the state of ionization
of a given group
Figure 4 The Henderson Hasselback Equation
Charged state of amino acids at neutral pH
The correct structure to represent an individual amino acid at neutral pH is +NH3-CHR-COO-
But when the amino acid is part of a peptide chain, the α-amino groups and α-carboxylate groups are
linked as uncharged amide bonds
x-NH-CHR-CO-x
The value of pKa tells you where in the pH scale a group undergoes
deprotonation
A molecule can have several ionizable groups
Each group has its own pKa value
Value of a pKa depends on its chemical context
an amino acid will have a slightly different pKa when it is part of a
peptide chain
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Document Summary

Small molecules sugars, amino acids, nucleotides, carboxylic acid derivatives. Protein size is expressed in kilodaltons (kda: 1 dalton (da) = 1 g/mol (mass of h atom, 1 kda = 1000 g/mol. Myoglobin is 16. 5 kda small protein. P-glycoprotein is 170 kda large protein. Proteins are made of amino acids linear chains of amino acids linked by peptide bonds (type of amide bond) Each protein has: unique sequence of different amino acids, a well-defined size and structure. Proteins have diverse functions including: catalyzing reactions (enzymes) forming complex subcellular structures. Each amino acid has an amino group and a carboxylate group. Each amino acid has a different side chain r. 20 different amino acids are found in proteins. Condensation involves removal of h2o from the units being linked. Hydrolysis regenerates the original carboxylic acid and amino group. The c=o group of the amide is the point of weakness allowing h2o attack.

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