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Fundementals I
Lecture 11-12
8/15/08
(Miller)
Continuation of Polar, uncharged group. (remaining 4)
POLAR, UNCHARGED
Threonine (Thr, T)
Side chain contains:
 Hydroxyl Group (OH)
 2 Carbon atoms
Cysteine (Cys, C)
 2nd amino acid containing sulfur. Methionine (Met, M) is the other amino acid
containing sulfur.
 Is “1st cousin” to Serine (Ser, S). Same structure except Cysteine contains a sulfur
atom where Serine contains an oxygen atom.
Tyrosine (Tyr, Y)
 Similar to Phenylalanine. Difference is Tyrosine contains a Hydroxyl (OH) para
(directly across) from the carbon atom on which the ring is attached.
Histidine (His, H)
 Is a critical amino acid because the imidazole ring is a potential site for acid-base
function.
 Recall titration curve from 8/14 Lecture.
 Has the ability to attach a proton here:


Side chain has a pKa value of 6.4. 50% of Histidine’s side chains will be
protonated.
Inceasing the pKa to 7.4: This means only 10% of Histidine’s side chains will be
positively charged (or protonated). (Protons are lost by increasing the pKa)


Decreasing the pKa to 5.4 means that 90% of Histidine’s side chains will be
positively charged. (or protonated)
Comes from Log relationship between pH pKa and ratio of anion and protonated
forms of acids.
POLAR, ACIDIC, NEGATIVELY CHARGED
-Referred to as acidic because the side chain contains a carboxyl group.
Acid Functions-each have a pKa
Asparic acid (Asp, D)
 Hydroxyl group attached to beta carbon
Strongest acid
pKa=2
pKa=9
Weakest
acid
Weaker acid
pKa=4

The carboxyl group attached to the alpha carbon is a weaker acid than the
carboxyl group attached to the beta carbon because the amine group carries a
positive charge making it easier for the nearby carboxyl group to donate a proton.
 The amino group will be protonated up to a very high pH.
 Remember: the higher the pKa values, the less acidic.
Glutamic acid (Glu, E)
 Hydroxyl group attached to gamma carbon
Weakest
Acid
pKa=9
Strongest
Acid
pKa=2
Weaker
Acid
POLAR
Histidine (His, H)
 Hard to group because it depends on environment as to whether it is not charged,
partially charged, or fully charged.
 Buffer capacity
POLAR, BASIC (POSITIVELY CHARGED)
Lysine (Lys, K)
 Has a 4-carbon side chain with primary amine on the end, connected to the
epsilon carbon
 Side-chains utilized to crosslink and stabilize collagen molecules.
Arginine (Arg, R)
 3-carbon straight chain with guanido group on end. pKa=12
 Side-chains set up to be splendid hydrogen bond agents.
pKa=2
pKa=9
pKa=12
Other amino acids exist in other proteins: They are made during translation or after
translation. (not part of genetic capability) There is a chemical modification of the sidechain to convert it.
Hydroxylysine, hydroxyproline- collagen
Carboxyglutamate- blood-clotting proteins
Pyroglutamate- in bacteriorhodopsin
Phosphorylated amino acids- a signaling molecules
Hydroxylysine:
Take the Lysine side-chain and on the delta carbon, a hydroxyl group has been added.
(this is done by lysyl hydroxylase) Hydroxyl group has a great influence on type of
cross-link that will be formed between collagen molecules
Hydroxyproline:
Often called 4-Hydroxyproline because the hydroxyl group is placed on the fourth carbon
atom.
 Counted carboxyl carbon as 1
 Alpha carbon as 2
 Beta carbon as 3
 Gamma carbon as 4
 Hydroxyl group is placed on praline, giving it the name hydroxyproline.
Has an influence in stabilizing collagen molecules. Collagen is the extracellular matrix
protein. Original 20 amino acids sufficed as long as there were only single cell
organisms. Organisms need organs and cells to act in concert. (extracellular matrix
needed to hold cells together) These additional organisms were developed to contribute to
an extracellular matrix. Ocean sponge great example. The holes are where the cells were
and extracellular matrix is the resulting sponge. (very similar to collagen)
Thyroxine:
Derived from tyrosine
Citrulline:
Derived from arginine. Will see Citrulline and ornithine in nitrogen metabolism lecture
later.
Serotonin:
Derived from tryptophan.
Difference is: carboxyl group has been removed from tryptophan (been decarboxylated)
and a hydroxyl group has been added to form serotonin. Serotonin=feel good molecule.
After the following two steps are taken, melatonin results. Melatonin-sleep drug.
Acetylate- “to bring an acetyl group into”
Methylate- “to attach or substitute a methyl group”
methylate
acetylate
Amino Acids are weak polyprotic acids.
Polyprotic acid-has many protons (can give up protons)
KNOW THESE NUMBERS ! (understand them, too)
Alpha carboxyl group have a pKa of 2.
Alpha amino group have a pKa of 9.
Amino group pKa=2
Carboxyl group pKa=9
The amino group is protonated when there is a net charge of +1 and pH of 1.
Decreased hydrogen ion concentration. Carboxyl group gives away proton (titrated
away) resulting in a net charge of 0 and pH of 7. (the carboxyl group lost all of it’s
protons and now has a negative charge.) (amino group still protonated because
pka=9)
The amino group gives away proton resulting in a net charge of -1 and pH of 13.
(amino group is now left uncharged)
Normal environment for life is a pH around 7.4. Free amino acids at this pH will look
similar to the above example. Altering pH alters molecule.
Substances can take up and give up protons to and from water.
Alpha amino and alpha hydroxyl groups go away when proteins are made because they
are “tied up” in peptide linkage.
Arginine’s (Arg, R) guanido group has a pKa value of 12.5
Aspartic Acid (Asp, D) has a pKa side-chain of 3.9
Cysteine’s (Cys, C) side chain has a pKa of 8.3. (sulfur-sulfhydryl group can ionize here)
At pKa of 7.4 (physiological pH) about 10% would be protonated.
Glutamic Acid (Glu, E) has a pKa of 4.3
Histidine (His, H) has a pKa of 6.0 to 6.4, depending on the environment.
pKa of His goes up on the inside of a globular protein in a non-aqueous environment.
pKa of His goes down to 6 when out in the environment.
Need to know these numbers because when we see a polypeptide chain, we need to be
able to change the pH, look at the molecule and decide what the charge will be on the
chain versus the pH. Side chains are what “matters” because they are free. Also
important are the first amino group of the first amino acid and the carboxyl group of the
last amino acid.
The more basic side-chains:
Lysine (Lys, K) pKa=10.5
Serine (Ser, S) pKa=13
Threonine (Thr, T) pKa=13
Tyrosine (Tyr, Y) pKa=10.1
*Would you expect Serine to ever have an ionization in a physiological system?
No, because you have to lower the hydrogen ion concentration to 1× 10^-13 before half
of it will be ionized.
When we deal with globular proteins (serine proteases) there is a way to “pluck” the
proton off the side-chain and make it a very electronegative atom.
(there IS a way for Serine to be ionized at a pH of 7.4)- will cover later
All amino acids can be buffering systems.
Carboxyl group can be ionized over a pH range of to 2.5 (for pKa of 1) and another range
from the amino group around pKa=9. Both sections are buffering ranges.
Conjugated Acid-Base system: Titration of glycine (from Figure 4.7)
Acid
Cationic form
Base
Neutral
Buffers:
All buffers need is a conjugated acid-base system.
Anionic
From figure 4.8: Titration of Glutamic acid. Similar to the titration of Histidine.
Alpha Amino
Gamma Carbon
Alpha Carbon
Pka of Alpha Carbon: 2
Pka of Gamma Carbon: 4
Pka of Alpha Amino: 10
(Won’t be asked to do many calculations for class.)
What is the pH of a glutamic acid solution if the alpha carboxyl is ¼ dissociated?
Use: pH = pKa + log ([A-]/[HA]) Henderson Hasselbalch
Ans.
pH = 2 + log10 [1] / [3]
( ¾ undissociated. ¼ dissociated. )
pH = 2 + (-0.477)
pH = 1.523
Titration of lysine:
1 real strong acid
2 weak acids
Alpha amino group and the group of the side chain.
What is the pH of a lysine solution if the side chain amino group is ¾ dissociated?
Ans.
pH = 10.5 + log10 [3] / [1]
pH = 10.5 + (0.477)
pH = 10.977 = 11.0
*if it were only ¼ dissociated, the pH would be lower than 10.5
Reactions of Amino Acids: be aware of these but focus mainly on Schiff Base:
An amino acid is reacted with an aldehyde (and water is lost) to form a Schiff base.
Schiff base-nitrogen double bonded to Carbon. Nitrogen has two extra electrons.
Mechanism wereby collagen molecules will crosslink with each other forming Schiff
bases using a physiological aldehyde, like Fixing tissues. (Tissue can be fixed by
formaldehyde). Two physiological formaldehydes called: Lysol oxidase and alpha amino
adipic acid that allow collagen molecules to become joined by crosslinking mechanism.
Example: (From figure 4.9)
Cysteine Example: (from figure 4.11)
Two cysteine side-chains react to form Cystine.
Two disulfide bonds oxidized to join and form from cysteine and cysteine to form
cystine.
Oxidation in biochemistry usually means taking away a hydrogen atom. (take away
proton and it’s associated electron)
Have left: 2 protons and 2 electrons.
Have taken away the hydrogens.
Each sulfur atom has 1 electron left so the cysteins are joined at the sulfurs to form
cystine.
On Board: (same example)
(2 cysteine molecules)
(hydrogens have been taken away and the remaining
electrons on sulfur have made a bond) (1 cystine molecule)
This is the way globular proteins are crosslinked.
Most globular proteins have crosslinks derived from the side-chains of 2 cysteine
molecules to form cystine.
Two side-chains are linked together and the polypeptide chain is crosslinked.
Crosslink can be intramolecular or intermolecular.
*Rarely in biochemistry, oxidation can occur from only the loss of an electron.
All amino acids have stereochemistry.
Glycine is the only amino acid that is not chiral. (doesn’t have four independent
constituents)
L-Amino Acids predominate in nature and are used exclusively in ribosomal synthesis of
proteins.
There are other proteins that are synthesized by other organisms (other than primates).
Some of these organisms can use D-amino acids. But D-amino acids are incorporated by
non-ribosomal protein synthesis (such as fungi)
D,L-nomenclature developed by sugar chemists
R,S-nomenclature designated as a superior.
Serine with one chiral center:
L-Serine differs from D-Serine because two can be superimposed as mirror images.
(like your hands) Called chirality. (means “hands” in greek)
Biological systems in which we will operate use the L-form of the amino acids.
In protein synthesis, only the L-form can be used.
Alanine:
L-Alanine
S-Alanine
R,S-nomenclature: Look at molecule with the hydrogen atom behind you (pointing into
the paper). If the side-chains and substituents go counter-clockwise (major substituent to
lower substituent), then the form is an S-form.
Atomic weight rates major to lower. (Heavier to lighter)
Example: Alanine
Nitrogen heavier than Carbon
Carbon with oxygen is Heavier than Carbon with Hydrogen
Therefore:
1. Amino group
2. carboxyl group
3. methyl group
S-Alanine
R,S-system brought in because certain amino acids like Isoleucine and threonine have
two chiral centers.
Mirror Images:
Also known as “enantiomers”
Diasterioisomers:
All depends on the configuration of the chiral centers.
L-Threonine and D-Threonine are enantiomers.
L-Threonine and L-Allothreonine are diasteriosomers.
L-Allothreonine and D-Allothreonine are enantiomers.
If there are more than one chiral centers:
The formula for the number of isomers: 2^n
n= number of chiral centers
Phe, Tyr, Trp absorb at UV wavelengths
Absorbance at 280 nanometers (nm) is a good diagnostic method for looking at proteins.
You can also look at proteins at peptide bonds which absorb at 190 nm.
Trp absorbs strongly at 280 nm
Tyr absorbs at a little less
Phe absorbs at 250 nm
All 3 absorb highly at 190 nm
Collagen molecules contain little Tyr and Phe and no Trp.
Collagen molecules must be observed at lower nm.
Amino Acid analyses are done by:
 Ion exchange chromatography
 High-performance liquid chromatography (HPLC)
Column with stationary phase of something like sulfonated polystyrene. With Active
group being sulfonic acid (negatively charge). The material is placed in a column (tube)
and it is “fixed”. Take advantage of the ion exchange or the acid-base properties of the
amino acid. Place amino acids in a column with three types of amino acids:
Aspartic Acid
Serine
Lysine
All amino acids will initially be attached to this particular resin because they’ll all be put
on at a low pH where they’ll all be positively charged. Begin to raise the pH and the
sodium ion concentration so that the sodium competes with the amino acid for sites on
the stationary phase. As that occurs, the aspartic acid molecules begin coming off first
(loses it’s positive charge holding it to the resin more quickly) As time passes, Serine
begins to come off, and finally lysine comes off.
Aspartic Acid loses positive charge quickly. It takes a much higher pH and a higher
concentration of sodium to get arginine from the column.
Refer to figure 4.19
Chromatographic Fractionation: (shows peaks)
Amino Acid analysis
Does not tell you the sequence or the numbers of amino acids in the proteins.
It does tell you the ratio of amino acids to each other.
Ion Exchange chromatography:
Exchanging the positively charged amino acids with sodium ions (positively charged).
Sodium ions knock off amino acids from column based upon overall positive charge.
Most positively charged amino acids (with highest pkas and more amino groups) will
come off later.
Histidine, ammonia, lysine, arginine. (Have extra positive charges in side-chains.)
Other amino acids have no positive charges in side chains and some are even negatively
charged.
Reversed phase chromatography
Derivitize each amino acid with a large hydrophobic group
Instead of an ionic polar column, the column is hydrophobic with no charge at all.
The molecules will absorb to the column based on their hydrophobicity.
(all amino acids will have some hydrophobic characteristics because they have been
derivitized with large hydrophobic molecule)
If amino acid is very hydrophobic, they will be taken off as gradient is changed from all
water to hydrophobic solvent.
0 hydrophobe to 100% hydrophobic.
Amino acids will be stuck while it is all water, but as solution gets more hydrophobic,
amino acids will leave the stationary environment.
Hydroxyproline is a very prominent amino acid.
Reversed phase chromatography can be completed in 35-45 minutes and in very small
quantities.
Ion exchange column takes hours and larger quantities.
Leucine is most prominent amino acid.
Least prominent is Tryptophan.
Nonpolar amino acids establish the folding pattern of globular proteins.
Globular proteins fold back on themselves and seek to hide from water.
Parts of the protein that are highly endowed with hydrophobic amino acids guide and
derive that process.
Polar, charged and uncharged amino acids establish the folding of fibrous proteins.
Fibrous proteins remain elongated and love water. Have more hydrophilic amino acids
than globular protein.
Amino acids like aspartic, glutamic, lysine, arginine are common in fibrous proteins.
Promote specific interactions
Histidine
Serine
Lysine
Asparagines
Cysteine