Practicals and tutorials Flashcards
Beer lambert law
A=E x c x l A: absorbance E: extinction coefficient (for the substance being measured) c: concentration of absorbing substance l: path length (use 1)
Formula for absorbance
Absorbance=log base 10 (transmittance in blank solution/transmittance in test solution)
Reaction catalysed by chymotrypsin
Catalyses hydrolysis of GPNA to N-glutaryl-L-phenyl alanine and p-nitroaniline. Latter absorbs at 410nm unlike the reactant.
(basically product absorbs at a wavelength diff to reactant)
Rate of enzyme catalysis
V0=Vmax * ([S]/([S]+Km))
i.e. rate=V max * (substrate conc/(substrate conc+Km))
What is Vmax and Km?
Vmax: max rate
Km: substrate conc at which you get half of Vmax.
Enzyme kinetics prac method summed up
- you have various substrate concentrations
- at each substrate concentration you measure the change in absorbance over time
- set the wavelength to the wavelength that the product absorbs at (in this case 410 nm)
- at each substrate concentration, calculate initial rate (change in absorbance/time) i.e. GRADIENT of the graph of absorbance against time
- convert V0 to 1/V0 and [S] to 1/[S}
- Plot a graph of reciporcals (s on x axis)
- calculate Vmax and Km from . graph
What’s the point of lineweaver burk plot?
Can’t determine Vmax easily so plot substrate conc and V as reciprocals.
Vmax from lineweaver burk plot
Y intercept is 1/Vmax.
Km from l-b plot
x-intercept=-1/Km
Effect of competitive inhibitor on Km and Vmax
Vmax is unchanged
Km increases.
On plot: no change in y intercept, but x intercept is less negative i.e. -1/Km is closer to 0.
(basically, compare in terms of positive values)
Effect of non-competitive inhibitor on Vmax and Km
Vmax decreases
Km is unchanged
On plot: no change in x intercept but y intercept increases (1/V0 increases, which means Vmax decreases)
Effect of SDS on electrophoresis
- allows proteins to be separated only on the basis of size (not charge)
Electrophoresis in solution vs gel
Solution: separation based on charge (e.g. HbS and HbA)
Gel: separation based on size and charge
Effect of mercaptoethanol on electrophoresis
Reduces disulphide bonds between cysteine residues
Mutation in osteogenesis imperfecta
- Col1A1 encodes Alpha-1
- Valine changes to Cysteine
- Introduces steric hindrance as cysteine is a larger amino acid
- introduces kinks in collagen triple helix
- affects the strength of the fibre
- hydroxapatite crystals don’t lay down on collagen properly–>compromises strength of bone–>fractures in long bones
Structure of collagen type 1
2 alpha 1 chains
1 alpha 2 chain
Why is osteogenesis imperfecta dominant not recessive?
- phenotype results from the loss of strutural integrity (i.e. loss of valine) rather than introduction of disulphide bonds
- thus you only need one mutated alpha 1 chain per triple helix to cause the phenotype (as opposed to 2 needed for disulphide bridge formation)
- majority of the collagen fibres are affected, leading to a dominant phenotype
Effects of osteogenesis imperfecta
- weak bones, skeletal abnormalities
- problems with eyes, teeth, skin and ears
Prenatal diagnosis of osteogenesis imperfecta
- CVS or amniocentesis
- PCR of the desired region of the gene (as you know where the mutation was in previous child).
- Hybridisation assay at high temperature and low ionic concentration so that you can detect region where there’s no compelmentarity
- if mutation at restriction enzyme recognition site, the enzyme will only cleave the WT not mutated
Uses of acyclovir
- treating immunocompromised people
- Zovirax: herpes cold sores (OTC)
- Varicella zoster infections (prescription)
Viruses in herpes virus group
DOUBLE STRANDED DNA VIRUSES characterised by their lATENCY (ability to induce life long infection) Herpes simplex (HSV types 1 and 2) EBV CMV Varicella zoster (VSV) Human herpes virus 6 (HHV6)
Structure of acyclovir
Guanosine analogue
How many CK isoenzymes?
3
MB in myocardium
15% of total CK
85% is MM
Separation of CK isoenzymes using electrophoresis
MM migrates fastest towards -ve electrode
