Biochem midterm

What are the three main types of macromolecules studied in biochemistry?

The 3 main types of macromolecules are proteins, made up of chains of amino acids.Polysaccharides- made up of simple sugars

Nucleic acids- made up of chains of nucleotides

Define polypeptide and oligopeptide, and state how they differ.

Polypeptide- long chains of amino acids

Oligopeptide- short chain of amino acids

What type of bond links amino acids in proteins?

Peptide bons linked amino acids in proteins

What is the average molecular weight of an amino acid before and after peptide bond formation?

The average weight is 128-18=110

How can you estimate the number of amino acids in a protein using molecular weight?

Dividing the molecular weight by 110

What are the two functional groups common to all amino acids?’

An amino group and a carboxylic acid group

What gives each amino acid its unique properties?

The functional R group that varies throughout amino acids

Describe what happens during condensation and hydrolysis reactions in peptide formation

During condensation there is a removal of H2O from the units being linked, hydrolysis regenerates the original carboxylic acid and amino that was lost, The double C=O bond is an easy site for the H2O to attack because it is a point of weakness.

What is the mass unit used to express protein size, and how is it related to grams per mole?

The mass unit to express protein size is kDa which is a kilodalton. 1Da=lg/mol 1kDa=1000g/mol

If myoglobin is 16.5 kDa, approximately how many amino acids does it contain?

16.5kDa=16,500 g/mol  

 # of amino acids= 16,500/110=150

What is the α-carbon in an amino acid, and why is it important?

The alpha carbon is the central carbon atom of the amino acid, the importance of this is that it is the central backbone of the atom

How many natural amino acids are there

20

Five main amino acid property groups

Very non-polar, moderately non-polar, polar but uncharged, polar and positively charged, polar and negatively charged

what is electronegativity

the tendency for an atom to attract electrons

What kind of bonds form between atoms of similar electronegativity

non-polar

What kind of bonds form between atoms of very different electronegativities 

Polar bonds 

Which amino acids have very non-polar side chains

alanine, leucine, valine, isoleucine, phenylalanine, methionine

Which amino acids are moderately non-polar?

glycine, cytesine, proline, tyrosine, tryptophan

Which amino acids are polar but uncharged? 

serine, threonine, glutamine, aspargnine

which amino acids are polar but positively charged (basic)?

arganine, histidine, lysine

which amino acids are polar but negatively charged?

aspartate, glutamate

what makes an amino polar but uncharged?

they form hydrogen bonds but they do not ionize at pH 7, so they cannot become protonated or deprotonated

What is a hydrogen bond donor?

A hydrogen covalently bonded to an electronegative atom (N,O)

What is a hydrogen bond acceptor? 

An electronegative atom with a lone pair of electrons (N,O,F)

How strong is a hydrogen bond compared to a covalent bond?

5-10% as strong

Are hydrogen bonds directional?

yes, they are, straight means stronger bond

what functional groups are found is Arg and Glu?

A carboxylic acid that is deprotonated at pH 7

What is a zwitterion?

A molecule with a positive and negative charge but a net charge of zero ( dipole ion, amino acids at pH 7)

what are the amino acids with ionizable side chains?

cysteine, tyrosine, glutamate, aspartate, lysine, histidine, arganine

What happens to Arg, His, and Lys side chains when deprotonated vs protonated?

when protonated they have a charge of +1 when deprotonated they are neutral (charge of zero)

What happens to to Asp, Glu, Cys, and Tyr side chains when deprotonated?

When deprotonated they have a charge of -1, when protonated they have a charge of zero

How does the pKa of an amino acid change when it’s part of a peptide chain?

It shifts slightly depending on the chemical environment of the peptide.

When pH = pKa, what percentage of the group is protonated vs deprotonated?

50% protonated, 50% deprotonated.

If the pH is one unit below the pKa, is the group mostly protonated or deprotonated?

Mostly protonated.

If the pH is one unit above the pKa, what is the ionization state?

Mostly deprotonated.

Do groups that ionize on oxygen or sulfur become positive when protonated?

No, they are neutral when protonated and negative when deprotonated.

Do groups that ionize on nitrogen become neutral or positive when protonated?

Positive when protonated and neutral when deprotonated.

What is the charge on the side chain of Aspartate at pH 5.0?

-1

What are the two main steps in amino acid analysis?

Separation and detection.

In partition chromatography, what are the stationary and mobile phases?

Stationary phase = polar solid (e.g., silica); mobile phase = nonpolar solvent.

In thin-layer chromatography (TLC), how is relative mobility (Rf) calculated?

A: Rf=XBXFR_f = \frac{X_B}{X_F}Rf​=XF​XB​​ (distance moved by compound ÷ distance moved by solvent).

Which type of amino acids have lower Rf values in TLC?

More polar amino acids.

How are amino acids detected after chromatography?

By using ninhydrin or fluorescamine reactions.

What color does ninhydrin produce with most amino acids?

Purple

What color does ninhydrin produce with proline?

yellow

What property does ion exchange chromatography separate molecules by?

charge

What type of stationary phase does a cation exchanger contain?

Negatively charged groups that bind positive molecules.

What type of stationary phase does an anion exchanger contain?

Positively charged groups that bind negative molecules.

How can amino acids be eluted from the resin?

By increasing salt concentration (e.g., NaCl) or changing the pH.

What is “elution volume”?

The volume of buffer required to move an amino acid through the column.

How are amino acids identified in ion exchange chromatography?

By comparing their elution volumes to standards (e.g., alanine or leucine).

What does ion exchange chromatography separate molecules by?

net charge

What type of resin is used in cation exchange chromatography?

A negatively charged resin that binds positively charged (cationic) molecules.

What type of resin is used in anion exchange chromatography?

A positively charged resin that binds negatively charged (anionic) molecules.

What determines how tightly an amino acid or protein binds to the column?

The magnitude of its net charge — larger charge = stronger binding.

How can proteins be eluted (released) from the column?

By increasing Na⁺ concentration (salt) or changing the pH to alter charge.

At pH 7, what percentage of proteins are typically negatively charged?

About 65% of all proteins.

In ion exchange, what does the elution volume represent?

The volume of buffer needed to move a molecule from top to bottom of the column.

What does affinity chromatography separate molecules based on?

Specific binding interactions between a protein and a ligand.

How are proteins eluted in affinity chromatography?

By adding a high concentration of salt or free ligand to compete for binding.

What is a “tag” in protein purification?

A small peptide or protein added to the target protein to help purify it through affinity chromatography.

In IMAC (Immobilized Metal Affinity Chromatography), what binds to the metal ions (Ni²⁺ or Co²⁺)?

Histidine residues in a His-tag.

What is the role of imidazole in IMAC?

It competes with His residues, displacing the His-tagged protein from the metal resin.

Why might a His-tag be removed after purification?

Because it can alter the protein’s properties or function.

What property does gel filtration separate proteins by?

ize (molecular weight

Which proteins elute first in gel filtration — large or small?

Large proteins elute first because they can’t enter the pores.

What is elution volume (Ve)?

The amount of buffer needed to move a protein through the column.

What kind of relationship exists between elution volume and log molar mass?

A linear relationship with a negative slope.

What does electrophoresis separate molecules by?

Movement in an electric field — based on charge, size, and shape.

What kind of gel is used for proteins?

Polyacrylamide (5–15% polymer, 90–95% water).

What dye is commonly used to visualize proteins in gels?

Coomassie Brilliant Blue.

What does SDS do to proteins?

Unfolds (denatures) them and gives them a uniform negative charge.

What property determines separation in SDS-PAGE?

Size only — smaller proteins move faster.

Why does SDS give proteins a uniform charge?

SDS coats them with negative sulfate groups, overriding their native charges.

How is molecular weight estimated in SDS-PAGE?

By comparing protein band positions to a protein ladder of known sizes.

What is the isoelectric point (pI)?

The pH at which a protein’s net charge = 0.

What happens to a protein in a pH gradient during IEF?

It moves until it reaches the pH where its net charge is zero, then stops.

At high pH, proteins are generally (protonated/deprotonated) and have what charge?

Deprotonated and negatively charged.

What two techniques are combined in 2D electrophoresis?

Isoelectric focusing and SDS-PAGE.

What does mass spectrometry measure?

the mass-to-charge ratio of ionized protein particles.

How does the time of flight relate to protein size?

inversely, Larger proteins move slower, taking longer to reach the detector.