BIOC12 LEC 3

Question 1

slide 5: what are the 5 biologically active molecules**

Answer 1

refer to slide

Question 2

slide 6: what are the 21st and 22nd amino acids

Answer 2

21st amino acid selenocysteine which contains selenium in place of sulfur
(found in a few proteins (has codon)
* 22nd pyrrolysine (archaebacteria: has codon)

Question 3

what amino acids have ionizable side chains

Answer 3

lysine,
arginine, histidine, aspartate, glutamate, cysteine, tyrosine

(do this by giving off or taking on protons when the pH is altered)

Question 4

when does the amino acid donate a proton

Answer 4

When the pH of the solution is below the pka the protonated form predominates and the amino acid is then a true acid that is capable of donating a proton

Question 5

when does the amino acid act as a proton acceptor

Answer 5

When the pH of the solution is above the of the ionizable group, the unprotonated form of that group predominates and the amino acid exists as the conjugate base, which is a proton acceptor

Question 6

sl 13: describe the histidine slide

Answer 6

the first (1.8) represents the ionization of the and the most basic value (9.3) represents the ionization of the group. The middle (6.0) corresponds to the deprotonation of the imidazolium ion of the side chain of histidine*

Question 7

what is pI and describe the migration

Answer 7

Isoelectric point (pI) = pH at which net charge
on an amino acid (or protein) is zero.

if placed on electric field at pH below its pI, it
would carry a net positive charge and migrate
toward cathode. At a pH above its pI, it would
carry a net negative charge and would migrate
toward anode

Question 8

how do you determine the pI for amino acids with 3 pKas

Answer 8

to determine pI for an amino acid with
three ionizable groups:
– add the two pKa that lie on either side of no
net charge

Question 9

how can you calculate the pH of a weak amino acid

Answer 9

the henderson haselbatch equation

Question 10

pKa values of ionizable side chains in
free amino acids can differ from those
in the amino acid chain of a protein

Answer 10

remember

Question 11

α-amino and α- carboxyl
groups exert a weaker influence on
neighboring side chains
– also the position of the side chain in the 3D
structure of the protein can affect its pKa

Answer 11

remember

Question 12

slide 21: how do amino acids link

Answer 12

Synthesis begins with the N-terminal amino acid—almost always methionine (Section 22.5)—and proceeds sequentially toward the C-terminus by adding one residue at a time.

Question 13

what are linked amino acids are called within the
amino acid chain

Answer 13

residues

Question 14

what is hydropathy

Answer 14

relative hydrophobicity/hydrophilicity of amino
acids
* usually measured by calculating tendency of
amino acid to prefer a hydrophobic environment
over a hydrophilic environment
* range from highly hydrophobic to weakly polar to
highly hydrophilic
* positive numbers on the scale usually refer to
hydrophobic side whereas the negative number
are the hydrophilic scale

Question 15

what is Fractionation

Answer 15

Fractionation is a separation process in which a certain quantity of a mixture (of gasses, solids, liquids, enzymes, or isotopes, or a suspension) is divided during a phase transition, into a number of smaller quantities

Question 16

how does salting out with ammonium sulphate work

Answer 16

enough ammonium sulfate is mixed with the solution of proteins to precipitate the less soluble impurities, which are removed by centrifugation. The target protein and other, more soluble proteins remain in the fluid, called the supernatant fraction. Next, more ammonium sulfate is added to the supernatant fraction until the desired protein is precipitated.

*the more salt gets protein of interest

Question 17

what is Column chromatography (overview)

Answer 17

A cylindrical column is filled with an insoluble material such as substituted cellulose fibers or synthetic beads. The protein mixture is applied to the column and washed through the matrix of insoluble material by the addition of solvent. As solvent flows through the column, the eluate (the liquid emerging from the bottom of the column) is collected in many fractions, a few of which are represented in Figure 3.11a. The concentration of protein in each fraction can be determined by measuring the absorbance of the eluate at a wavelength of 280 nm

Question 18

how does Column chromatography work

Answer 18

A mixture of proteins is added to a column containing a solid matrix. Solvent then flows into the column from a reservoir. Washed by solvent, different proteins (represented by red and blue bands) travel through the column at different rates, depending on their interactions with the matrix. Eluate is collected in a series of fractions, a few of which are shown. (b) The protein concentration of each fraction is determined by measuring the absorbance at 280 nm. The peaks correspond to the elution of the protein bands shown in (a). The fractions are then tested for the presence of the target protein

Question 19

how does ion exchange chromatography work

compare this understanding with slide 33

Answer 19

In ion-exchange chromatography, the matrix carries positive charges (anion exchange resins) or negative charges (cation-exchange resins). Anion-exchange matrices bind negatively charged proteins, retaining them in the matrix for subsequent elution. Conversely, cation-exchange materials bind positively charged proteins. The bound proteins can be serially eluted by gradually increasing the salt concentration in the solvent. As the salt concentration is increased, it eventually reaches a concentration where the salt ions outcompete proteins in binding to the matrix. At this concentration, the protein is released and is collected in the eluate. Individual bound proteins are eluted at different salt concentrations, and this fractionation makes ion-exchange chromatography a powerful tool in protein purification

Question 20

how does size exclusion/gel filtration

Answer 20

Gel-filtration chromatography separates proteins on the basis of molecular size. The gel is a matrix of porous beads. Proteins that are smaller than the average pore size penetrate much of the internal volume of the beads and are therefore retarded by the matrix as the buffer solution flows through the column. The smaller the protein, the later it elutes from the column. Fewer of the pores are accessible to larger protein molecules.

Question 21

how does Affinity chromatography is the most selective type of column chromatography

Answer 21

It relies on specific binding interactions between the target protein and some other molecule that is covalently bound to the matrix of the column.

As a mixture of proteins passes through the column, only the target protein specifically binds to the matrix. The column is then washed with buffer several times to rid it of nonspecifically bound proteins. Finally, the target protein can be eluted by washing the column with a solvent containing a high concentration of salt that disrupts the interaction between the protein and column matrix

Question 22

what is electrophoresis

Answer 22

which separates proteins base on migration in
an electric field (size and charge)

Question 23

all proteins are coated with SDS (detergent) giving all
proteins a net what charge

and what region they bind to

whats the binding ratio

Answer 23

net negative charge

• binds to the hydrophobic regions of proteins

because of the way SDS binds (one molecule per every two
residues) same charge to mass ratio

Question 24

what is the name and purpose of the reducing agent for sds page

Answer 24

also requires a reducing agent Beta mercaptoethanol or
dithiothreitol to break disulphide bridges