Friday, November 27, 2009
Thursday, November 19, 2009
UNIT 1: PROTEIN STRUCTURE AND FUNCTION (Chapters 1-4)
ASSIGNMENT NO. 1: WHAT IS THE IMPORTANCE OF BIOCHEMISTRY IN PHARMACY?
Minimum: 500 words, Maximum: 1200 words. Submission Date: 01 February 2010 in class
Typed using Times New Romans, Font size 12pt and Line spacing of 1.
On top of first page mention the Surname & Initials, Student Number, Course Code, Assignment One: What is the importance of Biochemistry in Pharmacy? … and then start with your assignment(don’t make a traditional cover page with your personal details, just go on and write)
Proteins are very important molecules in our cells. They are involved in virtually all cell functions. Each protein within the body has a specific function. Some proteins are involved in structural support, while others are involved in bodily movement, or in defense against germs. Proteins vary in structure as well as function. They are constructed from a set of 20 amino acids and have distinct three-dimensional shapes. Below is a list of several types of proteins and their functions.
Protein Functions
1. Antibodies - are specialized proteins involved in defending the body from antigens (foreign invaders). One way antibodies destroy antigens is by immobilizing them so that they can be destroyed by white blood cells.
2. Contractile Proteins - are responsible for movement. Examples include actin and myosin. These proteins are involved in muscle contraction and movement.
3. Enzymes - are proteins that facilitate biochemical reactions. They are often referred to as catalysts because they speed up chemical reactions. Examples include the enzymes lactase and pepsin. Lactase breaks down the sugar lactose found in milk. Pepsin is a digestive enzyme that works in the stomach to break down proteins in food.
4. Hormonal Proteins - are messenger proteins which help to coordinate certain bodily activities. Examples include insulin, oxytocin, and somatotropin. Insulin regulates glucose metabolism by controlling the blood-sugar concentration. Oxytocin stimulates contractions in females during childbirth. Somatotropin is a growth hormone that stimulates protein production in muscle cells.
5. Structural Proteins - are fibrous and stringy and provide support. Examples include keratin, collagen, and elastin. Keratins strengthen protective coverings such as hair, quills, feathers, horns, and beaks. Collagens and elastin provide support for connective tissues such as tendons and ligaments.
6. Storage Proteins - store amino acids. Examples include ovalbumin and casein. Ovalbumin is found in egg whites and casein is a milk-based protein.
7. Transport Proteins - are carrier proteins which move molecules from one place to another around the body. Examples include hemoglobin and cytochromes. Hemoglobin transports oxygen through the blood. Cytochromes operate in the electron transport chain as electron carrier proteins.
Chapter 1: Amino Acids
Titration of Aspartate with Hydroxide
On the left in both the chemical reaction and the titration curve, you should imagine that aspartic acid is in a very acidic solution at a pH of about 0. As you move from left to right accross the page, you are adding hydroxide to the solution. This increases the hydroxide concentration and decreases the hydrogen ion concentration. Note that aspartate loses protons as you move from left to right. At the first pKa, the alpha-carboxyl dissociates. At the second pKa, the R-group carboxyl dissociates, At the third pKa, the alpha-amino dissociates.
Note that at each pKa, the solution is buffered. That is, it resists changes in pH as hydroxide is added. Also note, that the pI occurs where aspartate has no net charge.
The main objective of this section is "Given any amino acid you must be able to predict the isoelectric point." How do I start? How do I figure this out without a graph to look at?
For this course, there are only four categories of isoelectric points that you need to learn:
- amino acids without any charged R-group (alanine, glycine, ...)
- lysine and arginine
- aspartate and glutamate
- histidine
The isoelectric point (pI) is the pH at which an amino acid or protein has no net charge and will not migrate towards the anode or cathode in an electric field. The charges on any amino acid at a given pH are a function of their pKas for dissociation of a proton from the alpha-carboxyl groups, the alpha-amino groups, and the side chains (R-group). The pKa for the alpha-amino groups and the alpha-carboxyl groups are about 2 and 10.
You start by having a very rough idea of the structure of the amino acid. What are the acidic groups and what are their pKas. Next, you try to visualize the amino acid fully associated with hydrogen and what the charge on the molecule would be. Next, you visualize removing hydrogen ions by titrating with hydroxide ions. You will remove hydrogen ions from the group with the lowest pKa first and, then from the next higher pKa. Eventually you reach the pI.
Example: Calculate the pI for Aspartate. Aspartic acid has an alpha-carboxyl, and alpha-amino and an R-Group that is a carboxyl group.
At very acidic pHs, the R-group is in the COOH form, the alpha-amino group is in the –NH3+ form and the alpha-carboxyl group is in the COOH form so aspartate has a net charge of +1
As we titrate with hydroxide ion, we remove hydrogen ions. They combine with hydroxide ions and become water. When we reach pH 2, the protons on half the alpha-carboxyl groups are removed. This is not the pI because half of the alpha-carboxyl have a negative charge but all of the alpha-amino groups –NH3+ have a positive charge and all of the R-Group (COOH ) have no charge. So, the net charge on the aspartate molecules is a positive 0.5.
As we titrate with more hydroxide ions, we reach a point half way between pKa1 and pKa2. At this pH, half of the protons have been removed from the two carboxyl groups and half of the carboxyl groups are not dissociated so the net charge on the carboxyl groups is a -1. The alpha-amino group is fully charged so it has a net charge of +1. The net charge on the aspartate molecules is 0. This is the pI
As we titrate with more hydroxide ions, we reach the pKa2, At this pH, all of the protons have been removed from the alpha-carboxyl group and half of the half of the protons have been removed from the R-group carboxyl groups. The net charge on the carboxyl groups is a -1.5. The alpha-amino group is fully charged so it has a net charge of +1. The net charge on the aspartate molecules is -0.5.
As we titrate with more hydroxide ions, we reach the pKa3, a point where all the carboxyl groups are dissociated and only half of the alpha-amino groups still have a positive charge. The net charge is a negative 1.5. We did not have to go this far to determine the pI but I thought it might be useful.
To review, you started with knowing that aspartate had three dissociable groups and the pKas for those groups. You know that as you titrate, the molecule will change as follows:
COOH, COOH, –NH3+ | at a pH below pKa1. The net charge is about +1. |
COO-, COOH, –NH3+ | at a pH between pKa1 and pKa2. Net charge is 0. |
COO-, COO-, –NH3+ | at pH above pKa2. The net charge is about -1. |
COO-, COO-, –NH2 | at a pH above pKa3, the net charge is about-2. |
The only neutral solution of aspartate must be when the pH is between pKa1 and pKa2. The pI is half way in between.pKa1 and pKa2
ACID-BASE PROBLEMS AND THE HENDERSON-HASSELBALCH EQUATION
A. A pharmaceutical molecule with antifungal properties is only active when deprotonated and negatively charged (A-). The protonated state (HA) is inactive. If the pKa of this drug is 10.0, (a) calculate the ratio of protonated to deprotonated compound at physiological pH (7.4). (b) Is this drug likely to be a useful pharmaceutical agent?
(a) calculate the ratio of protonated to deprotonated compound at physiological pH (7.4).
Since we are given both the pH and pKa of the compound, we can use the Henderson-Hasselbalch equation to solve for the ratio of [HA] to [A-].
pH = pKa - log([HA] / [A-])
log([HA] / [A-]) = pKa – pH
log([HA] / [A-]) = 10.0 – 7.4
log([HA] / [A-]) = 2.6
([HA] / [A-]) = 398.11
([HA] / [A-]) = 400 (correct sig figs)
The ratio of protonated (inactive) compound to deprotonated (active) compound is 400 to 1 at physiological pH.
(b) Is this drug likely to be a useful pharmaceutical agent?
Since the vast majority of the compound is in the inactive form at physiological pH, it is unlikely to be a useful pharmaceutical agent.* Ideally, most of the compound would be active in the body.
*However, if the active compound is highly potent, it is possible that a small fraction of active compound is sufficient for useful antifungal activity.
B.
Absorption of aspirin (acetylsalicylic acid, C9H8O4,) into the bloodstream occurs only when the molecule is in its conjugate base form.
(a) If a patient takes two tablets of aspirin (325 mg each), how many grams of aspirin are available for immediate absorption in the stomach? The pH of the stomach is 1.6, and the pKa of aspirin is 3.5.
Since we are given both the pH of the stomach and the pKa of aspirin, we can use the Henderson-Hasselbalch equation to solve for the ratio of [HA] to [A-].
pH = pKa - log([HA] / [A-])
log([HA] / [A-]) = pKa – pH
log([HA] / [A-]) = 3.5 – 1.6
log([HA] / [A-]) = 1.9
([HA] / [A-]) = 79
The ratio of protonated aspirin to its conjugate base is 79 to 1.
So one-eightieth (1/80) of the total aspirin taken will be in the conjugate base form and available for immediate absorption in the stomach:
2 x 325 mg x (1/80) = 8.75 mg
9 mg
(b) Would you expect more or less aspirin to be absorbed in the small intestine (pH ≈ 7.5) compared to the stomach? Briefly explain your answer (no calculation is required).
More aspirin will be absorbed in the small intestine. The higher pH in the intestine means that more aspirin will be in the conjugate base form and therefore available for absorption.
TEST YOURSELF
1.1 Which of the following is the smallest of all amino acids?
A).Glycine, B).Valine, C).Alanine, D).Serine
1.2 Which of the following amino acids can form a covalent bond called a disulfide bond?
A). C, B). K, C). M, D). Y
1.3 Which of the following amino acids is actually an imino acid?
A). Glutamate, B). Alanine, C). Proline D). Glycine
1.4 Which of the following amino acids is the largest of all amino acids?
A). E, B). Y, C). W, D). H
1.5 Which of the following amino acids is not positively charged?
A). H, B). K, C). D D). R
1.6 The three letter “asn” is for which amino acid?
A). Alanine, B). Aspartic acid, C). Asparagine, D). Arginine
1.7 Which of the following is a basic amino acid with a positive charge?
A). Lys, B). Glu C). Trp, D). Gln
1.8 Which of the following amino acids’ side chain is a single methyl group?
A). I, B). V, C). A, D). L
1.9 Which of the following amino acids can form an ionic bond?
A). E & V, B). D & K, C). R & Q, D). P & G
1.10Which of the following amino acids() are polar?
A). All of these, B). Tyrosine, C). Serine, D). Arginine
1.11Titration of Valine by a strong base reveals two pKa’s. The titration reaction occurring at pKa2(pKa2=9.62) is:
A. –COOH + OH- à -COO- + H2O C. –COO- + -NH2+- à -COOH + -NH2
B. –COOH + -NH2 à -COO- + -NH2+ D. –-NH3+ + OH- à -NH2 + H2O
1.12For amino acids with a neutral R group, at any pH below the pI of the amino acid, these amino acids in solution will have:
A). a net negative charge, B). a net positive charge, C). no charged groups, D). no net charge
1.13The peptide AEGAL has:
A). a S-S bridge, B). 5 peptide bonds, C). 4 peptide bonds, D). no free carboxyl groups.
1.14Charmaine was upset that her boyfriend dumped her over her best friend; so she decided to commit suicide by overdosing with Panado (pKa=3) and drinking it with lots of fresh milk (pH 6.7). She was admitted to the hospital but was later discharged without any serious complication. Drug tests revealed that most of the Panado remained in the stomach rather than being absorbed, can you explain these? (include calculations).
1.15You are working for a MERCK Pharmaceutics and the manager gives you a task of designing a drug to treat breast cancer and produce it in the form of a pill. The drug must have an absorption success of atleast 60% in the stomach (pH=2), which is where it must be in unionized form. What will the pKa of your drug be?
1.16A student titrates acidified Lysine using NaOH to completed all of its titrable groups. Draw the titration curve of Lysine showing the state of these Ionizable groups at each point and also calculate its pI.
1.17Hemoglobin is a protein responsible for transporting oxygen from the lungs to the tissue via the blood circulatory system. For it to be able to perform its function which of the following groups of amino acids will dominate the surface of hemoglobin?
A). A, V, F, L B). H, R, L, E C). Y, G, M, I D). W, D, A, V
1.18Insulin binds to insulin receptor on cell surfaces to be able to facilitate uptake of glucose by the cells. Which types of amino acids will dominate the surface of the Insulin receptor?
A). AVFL B). HRLE C). YGMN D). WDAV
1.19All the 19 amino acids have a chiral carbon center except for A). F B). G C). H D). W.
1.20Suppose you were a bacterium and had a choice of living in the small intestines (pH 8) or the stomach (pH 1). A new antibacterial drug (pKa = 2.5) has just been released into the market to be taken by humans. Which environment would choose to live in? The stomach or small intestine?
Chapter 2: Protein Structures
Joining Amino acids to the protein Primary Structure
We have already learned that the subunits of a protein are amino acids or to be precise amino acid residues. We know that an amino acid consists of a central carbon atom (Cα) and an amino group (NH2), a hydrogen atom (H), a carboxy group (COOH) and a side chain (R) which are bound to the Cα. A peptide bond is formed via covalent binding of the Carbon atom of the Carboxy group (COOH) of one amino acid to the nitrogen atom (NH2) of the amino group of another amino acid by dehydration: 
Figure 2.1: Peptide bond linking two amino acids A polypeptide chain is a chain of amino acid residues linked together by peptide bonds. The backbone of the polypeptide is given by the repeated sequence of three atoms of each residue in the chain: the amide N, the alpha Carbon Cα and the Carbonyl C. Rotations in the chain take place about the bonds in the backbone, whereat the peptide bond usually is unflexible (see Figure 2). The existence of an amino group (N-Terminal) at one end of the chain and a carboxy group (C-Terminal) at the other end designs a direction to the chain. Conventionally the beginning of a polypetide is its N-Terminal. 
Figure 2.2: Torsion (or dihedral) angles of the backbone
NB: See your prescribed textbook for characteristics of the peptide bond (Page 14).
Joining these amino acids together using a peptide bond gives rise to the linear polypeptide chain called the Primary Struture.
A). The primary structure of proteins
Drawing the amino acids
In chemistry, if you were to draw the structure of a general 2-amino acid, you would probably draw it like this:
However, for drawing the structures of proteins, we usually twist it so that the "R" group sticks out at the side. It is much easier to see what is happening if you do that.
That means that the two simplest amino acids, glycine and alanine, would be shown as:
Peptides and polypeptides
Glycine and alanine can combine together with the elimination of a molecule of water to produce a dipeptide. It is possible for this to happen in one of two different ways - so you might get two different dipeptides.
Either:
Or:
In each case, the linkage shown in blue in the structure of the dipeptide is known as a peptide bond. In chemistry, this would also be known as an amide linkage, but since we are now in the realms of biochemistry and biology, we'll use “the peptide bond”.
If you joined three amino acids together, you would get a tripeptide. If you joined lots and lots together (as in a protein chain), you get a polypeptide.
A protein chain will have somewhere in the range of 50 to 2000 amino acid residues. You have to use this term because strictly speaking a peptide chain isn't made up of amino acids. When the amino acids combine together, a water molecule is lost. The peptide chain is made up from what is left after the water is lost - in other words, is made up of amino acid residues.
By convention, when you are drawing peptide chains, the -NH2 group which hasn't been converted into a peptide link is written at the left-hand end. The unchanged -COOH group is written at the right-hand end.
The end of the peptide chain with the -NH2 group is known as the N-terminal, and the end with the -COOH group is the C-terminal.
A protein chain (with the N-terminal on the left) will therefore look like this:
The "R" groups come from the 20 amino acids which occur in proteins. The peptide chain is known as the backbone, and the "R" groups are known as side chains.You can determine the sequence of the primary structure by using several methods but please how to use enzymatic degradation to do so. We will use Trypsin and Cyanogen Bromide as examples in class.
B). The secondary structure of proteins
Within the long protein chains there are regions in which the chains are organised into regular structures known as alpha-helices (alpha-helixes) and beta-pleated sheets. These are the secondary structures in proteins.
These secondary structures are held together by hydrogen bonds. These form as shown in the diagram between one of the lone pairs on an oxygen atom and the hydrogen attached to a nitrogen atom:
Within the long protein chains there are regions in which the chains are organised into regular structures known as alpha-helices (alpha-helixes) and beta-pleated sheets. These are the secondary structures in proteins.
These secondary structures are held together by hydrogen bonds. These form as shown in the diagram between one of the lone pairs on an oxygen atom and the hydrogen attached to a nitrogen atom:
The alpha-helixIn an alpha-helix, the protein chain is coiled like a loosely-coiled spring. The "alpha" means that if you look down the length of the spring, the coiling is happening in a clockwise direction as it goes away from you.
Note: If your visual imagination is as hopeless as mine, the only way to really understand this is to get a bit of wire and coil it into a spring shape. The lead on your computer mouse is fine for doing this!
The next diagram shows how the alpha-helix is held together by hydrogen bonds. This is a very simplified diagram, missing out lots of atoms. We'll talk it through in some detail after you have had a look at it.
What's wrong with the diagram? Two things:
First of all, only the atoms on the parts of the coils facing you are shown. If you try to show all the atoms, the whole thing gets so complicated that it is virtually impossible to understand what is going on.
Secondly, There have no attempt whatsoever made to get the bond angles right. I have deliberately drawn all of the bonds in the backbone of the chain as if they lie along the spiral. In truth they stick out all over the place. Again, if you draw it properly it is virtually impossible to see the spiral.
So, what do you need to notice?
Notice that all the "R" groups are sticking out sideways from the main helix.
Notice the regular arrangement of the hydrogen bonds. All the N-H groups are pointing upwards, and all the C=O groups pointing downwards. Each of them is involved in a hydrogen bond.
And finally, although you can't see it from this incomplete diagram, each complete turn of the spiral has 3.6 (approximately) amino acid residues in it.
If you had a whole number of amino acid residues per turn, each group would have an identical group underneath it on the turn below. Hydrogen bonding can't happen under those circumstances.
Each turn has 3 complete amino acid residues and two atoms from the next one. That means that each turn is offset from the ones above and below, such that the N-H and C=O groups are brought into line with each other.
Beta-pleated sheets
In a beta-pleated sheet, the chains are folded so that they lie alongside each other. The next diagram shows what is known as an "anti-parallel" sheet. All that means is that next-door chains are heading in opposite directions. Given the way this particular folding happens, that would seem to be inevitable.
It isn't, in fact, inevitable! It is possible to have some much more complicated folding so that next-door chains are actually heading in the same direction. We are getting well beyond the demands of our syllabus now. But look at figure 2.8 (2nd edition) or figure 2.9 (3rd edition) in your prescribed text book.
The folded chains are again held together by hydrogen bonds involving exactly the same groups as in the alpha-helix.
C). The tertiary structure of proteins
What is tertiary structure?
The tertiary structure of a protein is a description of the way the whole chain (including the secondary structures) folds itself into its final 3-dimensional shape. This is often simplified into models like the following one for the enzyme dihydrofolate reductase. Enzymes are, of course, based on proteins.
Note: This diagram was obtained from the www.rcsb.org/pdb/ which is RCSB Protein Data Bank. If you want to find more information about dihydrofolate reductase visit this site.
There is nothing particularly special about this enzyme in terms of structure. I chose it because it contained only a single protein chain and had examples of both types of secondary structure in it.
The model shows the alpha-helices in the secondary structure as coils of "ribbon". The beta-pleated sheets are shown as flat bits of ribbon ending in an arrow head. The bits of the protein chain which are just random coils and loops are shown as bits of "string".
The colour coding in the model helps you to track your way around the structure - going through the spectrum from dark blue to end up at red.
You will also notice that this particular model has two other molecules locked into it (shown as ordinary molecular models). These are the two molecules whose reaction this enzyme catalyses.
What holds a protein into its tertiary structure?
The tertiary structure of a protein is held together by interactions between the the side chains - the "R" groups. There are several ways this can happen.
1. Ionic interactions
Some amino acids (such as aspartic acid and glutamic acid) contain an extra -COOH group. Some amino acids (such as lysine) contain an extra -NH2 group.
You can get a transfer of a hydrogen ion from the -COOH to the -NH2 group to form zwitterions just as in simple amino acids.
You could obviously get an ionic bond between the negative and the positive group if the chains folded in such a way that they were close to each other.
2. Hydrogen bonds
Notice that we are now talking about hydrogen bonds between side groups - not between groups actually in the backbone of the chain.
Lots of amino acids contain groups in the side chains which have a hydrogen atom attached to either an oxygen or a nitrogen atom. This is a classic situation where hydrogen bonding can occur.
For example, the amino acid serine contains an -OH group in the side chain. You could have a hydrogen bond set up between two serine residues in different parts of a folded chain.
You could easily imagine similar hydrogen bonding involving -OH groups, or -COOH groups, or -CONH2 groups, or -NH2 groups in various combinations - although you would have to be careful to remember that a -COOH group and an -NH2 group would form a zwitterion and produce stronger ionic bonding instead of hydrogen bonds.
3. van der Waals dispersion forces
Several amino acids have quite large hydrocarbon groups in their side chains. A few examples are shown below. Temporary fluctuating dipoles in one of these groups could induce opposite dipoles in another group on a nearby folded chain.
The dispersion forces set up would be enough to hold the folded structure together.
4. Sulphur bridges
Sulphur bridges which form between two cysteine residues.
If two cysteine side chains end up next to each other because of folding in the peptide chain, they can react to form a sulphur bridge. This is another covalent link and so some people count it as a part of the primary structure of the protein.
Because of the way sulphur bridges affect the way the protein folds, we will count this as a part of the tertiary structure.
C). The Quaternary structure of proteins
Quaternary Structure is the combination of two or more chains, to form a complete unit. The interactions between the chains are not different from those in tertiary structure, but are distinquished only by being interchain rather than intrachain.
Some proteins are composed of identical subunits (chains). A simple example is the dimer of HIV Protease.
Some proteins are composed of non-identical subunits (chains). A simple example is insulin which is made up of two chains, the alpha chain and the beta chain, linked by two disulfide bridges.
In our syllabus we will use Hemoglobin as an example of a protein with a quaternary structure.
To checkout the protein formations from primary struture to quartenary structure please click here.
The most important thing here is that as a pharmacist you should know the three dimensional structures of these protein so that you can design your drugs and medicines accordingly.
TEST YOURSELF
2.1 Choose whether the descriptions below match the
(A) Primary (B) Secondary, C)Tertiary or D) Quaternary Structure
(i) The structure formed by disulphide bridges between different parts of a polypeptide chain.
(ii) The highest level structure found in a protein with two or more peptide chains.
(iii) Folding due to electrostatic interactions between R-groups of the amino acids that make up the polypeptide chain.
(iv) The highest level structure found in a protein with a single peptide chain.
(v) The structure formed by covalent bonds between adjacent amino acid residues in a polypeptide.
(vi) The combination of two or more polypeptide chains.
(vii) The structure due to, amongst other things, van der Waals’ interactions between uncharged R groups.
(viii) The sequence of amino acid residues in a polypeptide chain.
(ix) The structure due to, amongst other things, hydrogen bonding between polar R groups.
(x) The pattern in which the polypeptide backbone folds and is stabilized by Hydrogen bonding between the N-H of one peptide and the C=O of another.
(xi) The alpha-helix
(xii) Always synthesized in the cell from the N-terminal end to the C-terminal end.
(xiii) The structure due to, amongst other things, ionic bonds between oppositely charged R groups.
(xiv) Β-pleated sheet
2.2 A polypeptide is cleaved into peptides by treatment with trypsin and cyanogens bromide, and the peptides are purified and sequenced. The sequences of the peptides are shown below.
Trypsin peptide
Cyanogen bromide peptides
T-1 GASMALIK
C-1 EGAAYHDFEPIDPRGASM
T-2 EGAAYHDFEPIDPR
C-2 TKDCVHSD
T-3 DCVHSD
C-3 ALIKYLIACGPM
T-4 YLIACGPMTK
Determine the primary sequence of the original polypeptide.
2.3
Structure of AspartameAspartame is an artificial sweetener found in Diet Coke, Coke Zero and most sugar-free soft drinks. Although it has roughly the same number of calories per gram as table sugar (sucrose), it is around 200 times sweeter. It was discovered by accident by James Schlatter, a chemist of G D Searle Co. in 1965, when he was testing an anti-ulcer drug. Its use in carbonated drinks was finally approved in 1983, following a decade-long battle against the objections of Dr. John Olney (a neuroscience researcher), James Turner (a consumer attorney) and investigations into the research practices of G D Searle. The objections were based on the amino acids Schlatter used to make Aspartame. Looking at its structure, which amino acids did James Schlatter use to make Aspartame?
2.4 Glutathione is a dietary supplement used as an antioxidant to help protect the body from many diseases and conditions. It is also used to treat infertility (difficulty getting pregnant), cancer, cataracts, and human immunodeficiency virus (HIV). Glutathione is used to detoxify various chemicals from the body.
Carefully observe the structure of glutathione and determine the three amino acids used to synthesize this useful drug. Give the name of this tripeptide.
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