Spectrophotometry and Enzymology L1

Question 1

why is absorbance a useful measurement

Answer 1

quick and easy
cheap
readily integrated into automation
reproducible
allows integration of multiple different assays which can be measured at different wavelengths

Question 2

what is the relationship between absorbance and concentration?

Answer 2

directly proportional

Question 3

relationship between conc and transmittance is

Answer 3

non-linear

Question 4

can calculate the absorbance from the transmittance

Answer 4

A=log10 1/T or A=2-log10 %T

Question 5

spectrophotometry

Answer 5

Spectrophotometry is a method toquantitatively measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution, generating an electrical current proportional to the intensity of the light.

Question 6

what is holmium oxide

Answer 6

holmium is a rare earth lanthanide element, it forms an oxide Ho2O3 which has a complex absorption spectra, with sharp, well defined peaks across the UV/visible range

Question 7

what are the different forms of spectrophotometry

Answer 7

reflectance
scatter
fluorescence
luminometry

Question 8

nephelometry

Answer 8

the detection of light energy scattered or reflected toward a detector that is not in the direct path of the transmitted light. more sensitive 1-10 mg/L, specialist equipment, measures light scatter at 90 degrees.

Question 9

turbidimetry

Answer 9

the measurement of turbidity by quantifying the degree of ‘attenuation’ of a beam of light of known initial intensity

Question 10

beer lambert law

Answer 10

concentration of a substance is directly proportional to the amount of light absorbed

Question 11

beer lambert equation

Answer 11

A= ecl

Question 12

what does the e stand for in beer lambert law

Answer 12

molar absorptivity (molar extinction coefficient)

Question 13

what does the c stand for in beer lambert law

Answer 13

concentration mol/L

Question 14

what does the I stand for in beer lambert law

Answer 14

Path length (cm)

Question 15

what is the path length

Answer 15

distance across cuvette (cm)

Question 16

factors affecting enzymes

Answer 16

pH
temp
type of buffer
cofactor
substrate
incubation time

Question 17

what enzymes do fixed-time methods use?

Answer 17

enzymes with high affinities (Low Km)

Question 18

how do fixed-time methods work?

Answer 18

measure substrate or product conc at 2 time points and calculate the difference then divide by time period to obtain rate

Question 19

what enzymes do continuous monitoring methods use?

Answer 19

enzymes with low affinities (high Km)

Question 20

how does a continuous monitoring method work?

Answer 20

reaction is monitored by continuously taking a rate measurement over a designated time period

Question 21

what are the advantages of a continuous monitoring method?

Answer 21

assess reaction progress, ensure true initial rate measurement obtained and identify any lag phase

Question 22

the exponential phase of the graph

Answer 22

optimal enzyme activity, the ideal point to be measured

Question 23

how to convert absorbance into rate

Answer 23

divide change in absorbance by the time period of measurement (mins) to give rate

Question 24

enzyme activity (u/l) equation

Answer 24

change in abs/min x 1,000,000 x total vol (ml) divided by e (L/mol/cm) x I (cm) x sample vol (ml)

Question 25

what does the michaelis menten equation describe

Answer 25

the effect of substrate concentration on the rate of enzyme catalysed reactions

Question 26

what is the michaelis-menten equation?

Answer 26

E + S —> K+/- 1 ES —> Kcat / K-2 E + P

Question 27

what is the steady state

Answer 27

when the ES is constant

Question 28

what is the equation for the initial reaction velocity at [S]

Answer 28

V=Vmax[S] / Km + [S]

Question 29

where is Vmax

Answer 29

slightly above the curve

Question 30

where is Km

Answer 30

Km is half the Vmax

Question 31

what is Km

Answer 31

Km is the substrate concentration at half-maximal velocity

Question 32

what is km a measure of

Answer 32

the strength of the ES complex

Question 33

what does a high Km indicate

Answer 33

weak binding

Question 34

what does a low Km indicate

Answer 34

strong binding

Question 35

what would we normally expect substrate concentration to be?

Answer 35

at least 10 Km

Question 36

what do enzyme catalysts do

Answer 36

they lower the activation energy for conversion of substrate to product

Question 37

what is referred to as the turnover number

Answer 37

Kcat

Question 38

lineweaver-Burk plot

Answer 38

reciprocals so can get a straight line
computers can do this

Question 39

competitive inhibitors

Answer 39

change the infinity of enzymes to the substrate

Question 40

non-competitive inhibitors

Answer 40

do not bind on the active site