Applied Biochemical Techniques

Question 1

explain how a spectrophotometer works and how it can be used to determine concentration of analyte

Answer 1

• light of continuous spectrum emerges from light source
• light passes through prism/grating which splits and refracts light into composite wavelengths
• sample is held in cuvette
• incident light enters into sample ad transmitted light emerges from sample
• intensity of transmitted light is detected by photodetector
• spectrophotometer then calculates amount of monochromatic light absorbed by sample
• intensity of light absorbed can be used to determine concentration of analyte

Question 2

describe each component parts of a spectrophotometer and the function of each part

Answer 2

light source - provides light that generates a continuous spectrum

cuvette - holds sample

prism/grating - splits and diffracts light into composite wavelengths

photodetector - detects intensity of transmitted light

Question 3

what is the difference between incident light and transmitted light?

Answer 3

• incident light enters into the sample
• transmitted light emerges from the sample

Question 4

what is absorbance (A)?

Answer 4

the amount of monochromatic light absorbed by a sample

Question 5

what is meant by the term λmax?

Answer 5

wavelength of light that is most strongly absorbed by the substance of interest

Question 6

why is it important to calibrate the spectrophotometer with a blank?

Answer 6

to take into account the natural absorbance of the solvent

Question 7

how is the effect of pH on the absorption spectra minimised?

Answer 7

prepare the sample in a suitable buffer solution

Question 8

explain why it is important to control the temperature of the sample during spectrophotometry?

Answer 8

temperature impacts on absorption measurements by causing expansion or contraction of solvent, changes to reaction rates

Question 9

what are the three factors of Beer-Lambert law that affect how much light is absorbed by a sample?

Answer 9

ϵ = molar absorptivity (how strongly a substance absorbs light of a given wavelength)

l = path length of cuvette (cm) (distance the light travels through the sample)

c = concentration of substance in a sample (moll-1)

Question 10

state the Beer-Lambert equation

Answer 10

A = ϵlc

Question 11

explain how you would use absorbance and concentration to determine the concentration of an unknown sample

Answer 11

• create a standard curve by measuring absorbance of known concentrations

use standard curve
- identify absorbance on y axis
- read along to line of best fit and read down to y axis
- mark this on graphs using dotted line

Question 12

explain a limitation of the Beer-Lambert law

Answer 12

high concentrations
- molecular interactions can take place
- causes changes to the position and shape of absorption bands
- limitation of BL law as it affects linearity of the relationship between sample concentration and absorbance
- low concentrations must be used

Question 13

state 2 applications of spectrophotometry

Answer 13

• enzyme activity
• quality/quantity of pharmaceuticals

Question 14

describe the basic principles of ion exchange chromatography

Answer 14

• matrix in column made up of polymer beads with a charge
• molecule mixture added to column and opposite charges attract
• molecules eluted by washing column with increasing concentrations of salt solution (NaCl)
• molecules move through column at rates determined by their charge

Question 15

explain how sodium chloride can be used to elute during ion exchange chromatography

Answer 15

molecule of interest has negative charge
- chlorine ions compete to bind with the matrix
- molecule with weakest negative charge elutes first

molecule of interest has positive charge
- sodium ions compete to bind with matrix
- molecule with weakest positive charge elutes first

Question 16

what charge does the column have if it is an ion exchange column?

Answer 16

anions are negative so the column must be positive

Question 17

what charge does the column have if it is a cation exchange column?

Answer 17

cations are positive so the column must be negative

Question 18

describe the basic principles of affinity chromatography

Answer 18

• column contains binding ligands that bind to the molecule of interest
• molecule mixture added to column
• target molecule becomes trapped (binds to ligand)
• elute using a ligand solution of higher concentration

Question 19

what is used for elution during affinity chromatography?

Answer 19

ligand solution (for example substrate)

Question 20

when separating enzymes using affinity chromatography, what property of the enzyme is being taken advantage of?

Answer 20

that it will bind to a substrate

Question 21

why does HPLC give a higher resolution compared to low pressure techniques?

Answer 21

sample spends less time in the column

Question 22

what type of gel is used to separate DNA fragments?

Answer 22

agarose

Question 23

what type of gel is used to separate proteins?

Answer 23

polyacrylamide

Question 24

what is the charge at the top of the tan next to the wells during DNA electrophoresis?

Answer 24

negative charge

Question 25

what would happen if you changed the pH if the buffer used during DNA electrophoresis?

Answer 25

change the charge on the phosphate group

Question 26

what part of DNA has a negative charge?

Answer 26

phosphate group

Question 27

what is the purpose of running a marker sample during electrophoresis?

Answer 27

to provide a standard to compare against/to determine DNA band size

Question 28

what could happen to the distance travelled by DNA fragments if the concentration of agarose is increases?

Answer 28

distance travelled would be less

Question 29

what property of proteins is being exploited during gel filtration?

Answer 29

size/molecular weight

Question 30

describe how gel alteration chromatography works

Answer 30

column contains a polymer (beads) that have pores/holes and retain molecules depending on their size

Question 31

what does SDS do in SDS-PAGE

Answer 31

it denatures proteins and coats their surface with negatively charged SDS ions

Question 32

what property of proteins is being exploited in molecular weight?

Answer 32

molecular weight

Question 33

name a stain that is commonly used which binds to proteins but not the gel?

Answer 33

coomassie blue

Question 34

what is meant by 'isoelectric point'?

Answer 34

the pH at which a protein has no charge

Question 35

what property of proteins is being exploited during isoelectric focusing?

Answer 35

each amino acid has its own isoelectric point and will migrate differently under the effect of different pH

Question 36

explain in detail how enzymes act as biological catalysts

Answer 36

- provide alternative mechanism for reaction - substrate binds to active site and enzyme facilitates reaction eg. stresses are brought to bear on chemical bonds in the substrate, makes chemical change more likely - interaction of enzyme and substrate reduces activation energy required for reaction to take place - more molecules can reach transition state therefore more product is formed

Question 37

describe 2 models that are used to describe enzyme activity

Answer 37

lock and key theory - for enzyme to catalyse reaction there must be an exact complementary fit between shape of enzyme's active site and shape of substrate molecule induced fit theory - conformation of an enzyme molecule is slightly altered when the substrate binds to the active site - enzyme folds around the substrate molecule

Question 38

explain how increasing temperature affects enzyme activity

Answer 38

- as temperature increases enzyme activity will initially increase as more molecules will have energy greater then activation energy - this continues until the optimum temperature where there is maximum activity - further increases in temperature will destabilise bonds within the enzyme leading to loss of tertiary structure. - this decreases enzyme activity as the active site will alter in structure - further increase in temperature will cause irreversible change to structure = enzyme denatured

Question 39

explain how pH affects enzyme activity

Answer 39

- bonds that hold substrate to active site depend of the correct charge on amino acid side chains - change the pH = alter charge on the side chains = affects binding - bonds that stabilise enzyme molecule depend on correct charge in amino acid side chains - change the pH= alter the charge on the side chains = affects the bonds - extremes of pH cause conformational change

Question 40

describe what the line in presence of a non-competitive inhibitor would look like in a graph?

Answer 40

- Max decreases because rate of reaction is slowed - proportion of enzyme molecules able to catalyse the reaction is diminished - Km is unchanged as affinity is not affected

Question 41

describe what the line in presence of a competitive inhibitor would look like in a graph?

Answer 41

- Km increases because inhibit compete s for active sites - means more substrate is needed to achieve half of Max - Vmax is unchanged because it is still achievable at high substrate concentrations