Mid-Term #1

What are small molecules?

1. Sugars

2. Amino acids

3. Nucleotides

4. Carboxylic acids derivatives

What are macromolecules?

1. Proteins, made as chains of amino acids

2. Nucleic acids, made as chains of nucleotides

3. Polysaccharides, made as chains of simple sugars

What is the size of most proteins?

10 000- 100 000 g/mol

What are Daltons?

1. unit of measurement for atomic mass; also known as atomic mass unit

2. 1 dalton = 1g/mol

What are proteins?

1. Chains of linked amino acids

2. The arrangement of amino acids in the chain determines the properties of function of each protein

3. Each protein has a unique sequence of amino acids and a well defined size and structure

What are the functions of proteins?

1. Catalyzing reactions

2. forming complex subcellular structures

What is a amine group?

NH2

What is a carboxyl group?

COOH

What is an amide group?

O=C-N

What is an ester group?

R-C=O-O-R

What is an amide bond?

In combination of an amine group and carboxyl acid

What are glycosidic bonds?

-C-O-C- links between sugars

What is an ester bond?

a bond formed by a condensation reaction between glycerol and a fatty acid

What is the basic structure of an amino acid?

An amine group, a carboxyl group, and a functional R-group.

What is a chiral center?

A chiral center is (usually) a carbon atom bearing four different substituents. This makes it so that the two are mirror images and nonsuperimposable. If there is one chiral center there are two enantiomers.

What are sterioisomers?

Molecules with the same structural formula but a different spatial arrangement of atoms

What are enantiomers?

a pair of molecules that exist in two forms that are mirror images of one another but cannot be superimposed one upon the other

What is a D-amino acid?

amino group on the right

What is an L-amino acid?

amino group on the left. This is how amino acids in proteins are found

What is a condensation reaction?

A reaction in which two molecules combine to form a larger molecule, producing H2O as a by product

What is a hydrolysis reaction?

1. a reaction in which a bond is broken by the addition of a water molecule

2. The C=O group of the amide is the point of weakness allowing water to attach

What is the equilibrium of hydrolysis?

1. Under standard biochemical conditions, the equilibrium of the reaction lies in the direction

of hydrolysis

2. For the reaction to move in the direction of

peptide bond synthesis, the carboxyl group

needs to be chemically modified or activated.

What are polypeptides?

a chain with many amino acids, usually a complete protein

What are oligopeptides?

a chain with a few amino acids, usually a fragment

What is the N-terminal amino acid?

The end with the uncombined amino group

What is the C-terminal amino acid?

The end with the uncombined carboxylate group

What are amino acid residues?

1. amino acids that are incorporated into a protein

2. The number of amino acid residues in a protein can be estimated by

dividing the molecular weight of the protein by 110

What is an alpha carbon?

The carbon adjacent to a functional group

What is the beta carbon?

the first atom of the side chain, the y-carbon is the second, etc

What is the Greek Alphabet?

Alpha, Beta, Gamma, Delta, Epsilon...

What are the amino acids with very non-polar side chains?

1. Ala, Val, Leu, Ile, Met, Phe

2. dominated by hydrocarbons

3. Hydrocarbons are non-polar and hydrophobic

What are amino acids with moderately non-polar side chains?

1. Glycine has single H atom as side chain, not

enough to be very non-polar

2. Cysteine contains the slightly polar SH

group

3. Proline is unique because the side chain

links to alpha-N as well as to alpha-C. The polar N

moderates the non-polar hydrocarbon.

4. Tyrosine has a single polar group that partly

offsets the very non-polar benzene ring.

Tryptophan behaves similarly.

What are amino acids with polar uncharged side chains?

1. Serine and threonine have side chains that include the polar hydroxyl group -

OH (simple alcohol)

2. Asn and Gln both contain the polar amide group

3. All four side chains act as good hydrogen bond donors or acceptors

What are hydrogen bonds?

1. Electrostatic attractions between a hydrogen bond doner and an acceptor

2.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

3. H-bonds are directional - stronger if donor and acceptor line up with one another

What are hydrogen bond doners?

Highly polar -OH

or -NH groups

What are hydrogen bond acceptors?

An acceptor is an

electronegative atom

with an available lone

pair of electrons, such

as O or N

What are positively charged side chains?

1. His, Lys and Arg

2. These side chains contain weak bases that gain H+ (become protonated) and so are positively charged in aqueous solution

at neutral pH

3. Charge makes them polar

What are negatively charged side chains?

1. Asp and Glu

2. Side chains have carboxylic acid groups R-COOH that lose H+

(become deprotonated) at neutral pH

3. When deprotonated these are described as carboxylate groups R-

COO-

4. Carboxylate groups are negative and also very polar

How to amino acids act as acids and bases?

1. The amino groups, the carboxylate groups, and the side chains or some amino acids can act as acids or bases

2. These groups will gain (protonate) or lose (deprotonate) H+ depending on

availability of H+ in solution

3. Each ionic functional group has a characteristic pKa which expresses the tendency to gain or lose H+

What is the Henderson-Hasselbalch equation?

pH = pKa + log [A-]/[HA]

What is the equation for ph?

pH = -log[H+]

What are the 7 amino acids with ionizable side chains?

1. Aspartate

2. Glutamate

3. tyrosine

4. cysteine

5. Arginine

6. histidine

7. Lysine

What is pKa?

-logKa

What does a low pKa mean?

strong acid

What does a high pKa mean?

weak acid

Why does alpha carboxylate have a lower pH?

Since it is located by the positive NH3 group, this position favor's deprotonation more than the side chains

How does pKa determine ionization?

1. As we raise the pH, [H+] becomes

less available, so deprotonation is

more likely to occur.

2. Each group undergoes a transition

as pH shifts from 0 to 14, starting ~1

unit below its pKa and almost

complete by ~1 unit above its pKa.

3. When pH = pKa, 50% of the group is

in the protonated form and 50% is in

the deprotonated form

If pH is one unit or more below the pKa...

the group is full protonated

If pH is one unit or more higher than pKa...

The group is fully deprotonated

If pH is equal to pKa...

50% deprotonated and 50% protonated

If pH is less than one unit away from pKa...

a calculation may be needed to

determine the exact state

How to we determine overall charge?

we multiply each fraction of protonated and deprotonated

with its associated charge

How can a functional group have a partial charge?

1. At pH 7, histidine is 76% deprotonated and 24% protonated

2. Molecules exchange H+ millions of times per second

3. A given His molecule is protonated 24% of the time (charge of +1) and

deprotonated 76% of the time (neutral)

4. Averaged over time, charge on His at pH 7 is: (0.24 x +1) + (0.76 x 0) = +0.24

What are buffers?

1. weak acids or bases that can react with strong acids or bases to prevent sharp, sudden changes in pH

2. The resulting pH will be close to the pKa of the buffer.

What are buffers in living cells?

bicarbonate ions, phosphate ion and phosphate

esters, all of which have pKa values close to 7.

What is amino acid analysis?

1. Separation of mixture into components

2. Detection of components of interest

- this can be qualitative (what is present_

-Quantitative (how much is present)

- Preparative (separated

components can be recovered for further

experiments)

What is seperation?

based on the different properties of the side chains, such as polarity or charge.

Separation is generally achieved by some form of chromatography.

What is detection?

based on chemical reactions that generate coloured or fluorescent amino acid

derivatives that can be seen and measured.

What is partition chromatography?

1. Stationary phase: particles of solid are chosen with a specific property, e.g. silica

gel has HO-Si-OH groups that can hydrogen-bond to polar

amino acids

2. Mobile phase:

Liquid solvent or buffer flows past the particles and is non-polar

3. Polar and charged amino acids will stick and move more slowly and non-polar will flow through

How are amino acids identified with chromatography?

1. Concentration of amino acids

is measured in each test tube and the results are graphed

2. To elute more polar amino acids, progressively polar

mobile phase is run through the column

3. Identified by their characteristic elution volume

What is thin layer chromatography?

In thin layer chromatography, a sample is spotted onto a silica plate, then carried upward by a solvent. The amino acids in the sample are separated by polarity: less polar lipids do not adhere to the plate as strongly and move farther up the plate.

What is the solvent front?

the distance moved by the solvent

What is the retention factor?

distance moved by pigment/distance moved by solvent (of TLC)

What compound is used to visualize amino acids?

1. ninhydrin which reacts with primary and secondary amines

2. Gives purple colour, 10^-8 moles detectable

3. Proline is yellow

4. Spray and heat

5. Colour intensity is proportional to quantity of amino acid, and can be measured

What is fluroscamine?

reacts with -amino N to give yellow fluorescence (10-10 mol detectable). When

illuminated with UV lamp, sample emits a yellow glow.

What is ion exchange chromatography?

1. A second type of liquid chromatography that separates molecules based on whether they have a negative or positive charge and based on how strong the charge is

2. Uses charged resins as stationary phase

3. Elution is completed 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

What is cation exchange chromatography?

Cation exchanger resins contain negative groups, which bind positive

molecules

What is anion exchange chromatography?

Anion exchanger resins contain positive groups, which bind negative

molecules

How are proteins separated from complex mixtures?

1. Proteins are derived from natural sources

such as microbial cultures, plants, or

animal tissues such as liver

2. The cells are broken open to release the

proteins into a solution or crude extract

3. Extracts may contain thousands of

different proteins

4. Separation by ion exchange is based on charge differences among proteins

• ~65% of all proteins are negatively charged at pH 7

What is affinity chromatography?

1. Ligands are covalently attached to the beads in the column

2. Proteins that have an affinity towards the

ligand bind tightly to it

3. Other proteins move faster down the

column and are eluted from the column

4. The bound proteins are eluted by the

addition of high concentration of salt

(weakens the binding between the ligand

and the proteins) or ligand (competes

with attached ligands)

What are tags?

A peptide or protein that binds a ligand with

high affinity and specificity that is fused to the gene

encoding target protein

What is metal affinity chromatography?

1. Clusters of His in a protein bind tightly to Ni2+ or Co2+ (ligands)

2. DNA encoding HIS tag then expresses 6-8 extra HIS residues to the target protein at the N or C terminus

3. Column is made up of chelating resin containing Ni2+

4. The bound protein is eluted by adding imidazole (structure similar to His side chain) to the buffer

5. High degree of purification in one step but can affect properties of the protein

6. Tag can be removed after isolation

What is gel filtration chromatography?

1. Separates on basis of size

2. Beads of polymeric gel - a loose network of

polymer with many water-filled pores.

3. Protein molecules can enter the pores if they fit and Larger proteins are excluded from the pores

4. Proteins of intermediate size may enter

some of the pores

5. Proteins separate by size; larger proteins elute first,

smaller proteins elute later

How is gel filtration used to measure the molar mass of proteins?

1. Measure elution volume of proteins of

known mass

2. Elution volume Ve is the volume of buffer

needed to move a protein from top to

bottom of column

3. Elution volume is a linear function of

log molar mass (negative slope)

4. Find antilog to determine the molar mass of unknown

5. Or you can use y-mx+b

What is electrophoresis?

1. the movement of charged particles in a fluid or gel under the influence of an electric field

2. Gels are normally made of a polymer of polyacrylamide

3. Separated proteins are visualized by adding a dye such as Coomassie blue which

binds to proteins

4. The rate of movement depends on size, shape, and charge

What can we estimate with

electrophoresis?

1. Does not contribute to purification as structure is

commonly affected by electrophoresis

2. Can visualize and characterize purified proteins

3. can be used to estimate:

• number of different proteins in a mixture

• degree of purity of a mixture

• isoelectric point

• approximate molecular weight

What is SDS-polyacrylamide gel electrophoresis?

1. SDS binds to the proteins and partially unfolds them into a similar rod shaped structure

2. The sulfate moieties of the SDS molecules confer a large

net negative charge on the protein swamping out the

native charge

3. uniform charges per unit size

4. Separation is based strictly on size, smaller moves faster than large ones

5. By comparing with the positions to which proteins of

known molecular weight (standards) migrates to in the gel,

the molecular weight of an unknown protein can be estimated

6. If a protein consists of multiple subunits, they are usually

separated by SDS-PAGE and shows up as multiple bands

What is isoelectric focusing?

1. Isoelectric focusing (IEF) is an electrophoretic technique for the separation of proteins based on their isoelectric point (pI).

2. The pI is the pH at which a protein has no net charge and thus, does not migrate further in an electric field.

3. At a high pH, the protein is deprotonated and move's towards the positive electrode (remove proton =more negative)

What is the isoelectric point?

the pH at which the molecule is electrically neutral

What is two-dimensional gel electrophoresis?

Proteins separate as spots based on their PI and molecular weight

1. Initially separated by isoelectric focusing (ie isoelectric points based on pH)

2. Second dimension they separate based on molecular weight using SDS electrophoresis

What is mass spectrometry?

1. A protein is vaporized by laser beam, yielding charged protein particles

2. Particles travel toward the detector

3. Velocity depends inversely on the mass of the particle (larger = slower)

4. The time of flight to the detector yields a very accurate mass measurement

5. Compare to data base

What is enzyme activity?

1. enzyme activity measures the number of moles of

substrate converted to product per unit time by the enzyme.

2. The total units of enzyme present in a solution

3. Theoretically, the amount of the target enzyme present in the mixture after

each purification step should remain constant.

4. Unit: 1 umol/min at 25 degrees

What is specific activity?

1. Ratio of enzymatic activity to the total amount of protein in a sample

2. The number of enzyme units per milligram of total protein (μmol min-1mg-1)

3. specific activity is a measure of

enzyme purity

4. Remains constant when pure

What happens once a protein is successfully isolated?

1. Further purification steps do not lead to an

increase in specific Activity

2. Only a single protein species is detected (e.g.

SDS-PAGE)

How do we investigate the structure of proteins?

1. What amino acids are contained in it

2. In what order or sequence do they occur

3. To do this we have to break the peptide bonds so they amino acids can be identified

Why is water not the best for hydrolysis?

1. Water attacks peptide bonds extremally slowly due to the neutral oxygen is a poor nucleophile

2. Although it has two lone pairs, the electronegative O is less willing to share them than with N or S

What is acid hydrolysis?

1. Done in 6M HCl at 110 degrees Celsius

2. Takes 24-72 hours

3. Tryptophan is destroyed in this process

What is base hydrolysis?

1. Done in 4M NaOH at 110 degrees Celsius

2. Takes 16 hours to complete

3. Some amino acids are destroyed in strong base

What happens when acid/base hydrolysis destroys the amino acids?

Also hydrolyses the amide bonds of Asn and Gln, converting them to Asp

and Glu respectively and releasing the amino group as ammonia

What is hydrolysis with digestive enzymes?

Proteases are enzyme with catalytic function and they can catalyze the hydrolysis of peptide bonds

What are nucleophiles?

1. an atom with a lone pair of electrons

available to share

2. Nucleophiles seek out other groups that are electron-deficient ("nuclei")-

electrophiles

3. Negative species or neutral species with a long pair of electrons

What is an electrophile?

1. A substance which can accept a pair of electrons

(electron pair acceptor)

2. Often compounds with double bonds or positive species

What can an atom with lone pairs do?

1. hydrogen bonds acceptor - attracts OH and NH

2. Base - it captures H+

3. Act as a nucleophile and share the lone pair with an electron deficient atom to make a new bond

What is nucleophilic subsitution?

incoming nucleophile X attacks target atom C to displace

leaving group Y which takes its bonding electrons away

What is nucleophilic addition?

A pi bond is removed and replaced with 2 new covalent bonds

What does a curly arrow represent?

Movement of an electron pair

Hydrolysis via nucleophilic subsitution

1. Water dissociates and exists in equilibrium of OH and H+ in water

2. The carbon on the C=O is electron deficient and can accommodate an incoming electron pair from OH-

3. The electrons in the double bond then temporarily move the O-

4. Since carbon can only accommodate 4 bonds, the N group leaves with the addition of H+ with the electrons and the double bond reappears

What is a leaving group?

A leaving group is a stable species that detaches itself from a molecule with its electron pair during the course of a reaction.

What is the transition state?

the complex formed as covalent bonds are being broken and re-formed during a reaction

What is the primary structure of a protein?

1. sequence of amino acids in a polypeptide chain

2. By convention the Nterminal amino acid is considered the start