Michaelmas week 5 Flashcards
Describe peptidoglycan briefly
It is the largest macromolecule in a bacterial cell
It consists of linear polysaccharide strands made of n-acetyl muramyl peptide and N-acetyl glucosamine linked by beta-1,4 glycosidic bonds
How does peptidoglycan get strength?
From peptide cross linking between different polysaccharide strands
What is peptidoglycan’s function?
To protect the bacteria against high internal osmotic pressure
Penicillin…
Inhibits peptidoglycan synthesis
Who found lysozyme and where?
Fleming, in tears
Where is lysozyme found?
Ruminants guts and egg white (stops bacteria invading eggs)
What is the difference between penicillin and lysozyme?
Lysozyme digests existing bacterial peptidoglycan by hydrolysing its polysaccharide backbone into disaccharides that still have peptide units attached
Bacteria lyse when their structural integrity is lost
What is more sensitive to lysozyme, gram positive or negative?
Positive because there is no protective membrane that can cover peptidoglycan
In our experiement what bacteria did we look at, was it gram +ve or -ve?
M. Luteus, gram positive
How can we look at lysozyme effects on M. Luteus?
At first M.Luteus suspension is opaque because it scatters light
When cell wall is removed light scattering decreases
Enzyme causes apparent drop in absorbance on spectrophotometer
Do light scattering measurement follow Beer-Lamberts law?
No, not like absorbance
Draw graph of attenuance vs. mg M. Luteus in assay
Graph od OD600 vs. time
(See notes for diagram)
OD600=?
Apparent absorbance 600
Why is the lysozyme curve exponential?
As walls are attacked they disintegrate into pieces that scatter much less light than the original cells
Fragments could be substrates for lysozyme but further digestion is invisible to the assay
Draw a graph of rate of change of OD600 vs enzyme micrograms in 3ml assay
(See notes for diagram)
What is lysozyme optimum pH?
Around 6
Describe lysozyme heat stability
What buffer do you heat it in?
If enzyme is heated for 10 minutes in a phosphate/citrate buffer, cooled and assayed it survives at 50,60 and 70 degrees
It loses half its activity at 80 and nearly all at 90
Why is lysozyme very stable?
Where is this a common feature?
It contains -S-S- bridges
This is a common feature in enzymes designed to work outside cells in an unfriendly environment
What happens if lysozyme is heated with DTT, what does this show?
DTT reduces disulphide bridge, enzyme killed
Shows heat stability is due to cross-linked cysteine residues
If you add DTT and SDS what happens?
SDS stops the protein unfolding so there is no aggregation
Draw results table for DTT and SDS
(See notes for diagram)
