Applications of Spectrophotometry Flashcards
What is meant by a coupled assay?
- The product of one enzyme reaction is used as the substrate of a second enzyme reaction
- The second enzyme reaction produces a detectable signal that can be measured
Explain how gamma-glutamyl p-nitroanilide can be used in an enzyme assay for gamma glutamyl transferase (GGT)
- GGT activity can be measured by its ability to convert gamma-glutamyl p-nitroanilide into p-nitroaniline
- P-nitroaniline absorbs at 410nm
- Higher the 410nm absorption the higher the GGT levels
Explain the enzymatic assays for cholesterol detection and quantification
- Cholesterol ester is converted into free cholesterol by cholesterol esterase prior to assay
- Free cholesterol oxidised by cholesterol oxidase
- Hydrogen peroxide is produced
- 4-aminoantipyrine plus phenol is catalysed by horse radish peroxidase (HRP) to form quinoneimine
- Quinoneimine is detected at 505nm
Which of the following amino acids has the highest extinction coefficient at 280nm?
a) A
b) C
c) P
d) W
e) Y
d) W
What is the formula for calculating the specific extinction coefficient at 280nm of a peptide from its amino acid constituents?
ε280 = nW x 5,500 + nY x 1,490 + nC x 125
Calculate the extinction coefficient at 280nm of the following peptide:
MWYWCAPYCWK
- ε280 = nW x 5,500 + nY x 1,490 + nC x 125
- ε280 = 3 x 5,500 + 2 x 1,490 + 2 x 125 = 19730 M^-1cm^-1
What is halochromism?
A substance that changes colour as the pH changes
(pH indicator)
What happens to phenolphthalein under basic pH conditions?
Turns pink / fuchsia
What is phenol red?
pH indicator dye used in tissue culture media
Explain the basis of phenol reds colour change at different pH
- Transition from yellow to red over a pH range of 6.2 - 8.2
- Above pH 8.2, phenol red turns pink / fuchsia
- Increased pH results in a yellow colour due to the loss of excess proton in ketone group
Explain how it is used as a pH indicator in cell culture
- Mammalian cells are neutral physiological pH
- A Bicarbonate-CO2 buffer system maintains physiological pH
- Bicarbonate increases pH while CO2 dissolves to form carbonic acid
- Cell waste products decrease pH
- Phenol red can be used to monitor the pH and health of tissue culture
- Red = healthy
- Yellow = unhealthy
Explain the biochemical basis of the Bradford test
- Coomassie G250 has absorption at 465nm
- It binds basic ( Arg, Lys, His) and aromatic (Tyr, Thr, Phe) side chains on proteins
- Coomassie changes to a blue colour and has an aborption of 595nm when bound to proteins
What happens to the UV-Vis spectrum of Coomassie blue when it binds to protein in solution?
Bathochromic Shift
(465nm to 595nm)
Explain the Limit of Detection (LOD)
LOD - the lowest concentration that can be reliably distringuished from background noise at the point at which the presence of the substance can be reliably confirmed with confidence
Explain the Limit of Quantification (LOQ)
LOQ - the lowest value of a signal that can be accurately measured and quantified with confidence
