Enzyme Activity-> Lysozyme

Features of lysozome

• small

• Not easilt destroyed

• Gram positive are more sensitive to it

  • have no outwer membrane to protect it

• Can be found with specrophotomer see the light scattering→ apparent absorbance

Experiement

• Assay lysozyme acitivty folloing the lysis of M.luteus

Check the light scattering properties of Micrococus lutes bacteria

1. cuvette add buffer

2. use as a blank for spectropphotomerer

3. Another one ass buffer and bacteria

4. mix well

5. Measure Apperent absoacne OD

6. Repeat with differen bacteria volumes

7. PLot a calibration curve

Relationship of the calibration curve

• Should be linear

Lysozyme activity assay

1. At room temperature

2. ass buffer and bacteria

3. put in spectomephtor

4. take reading

5. Add lysozme WUICKLY and mix

6. Put into spectrophotomerter reed attenuance

7. Take readings every 10 seconds

8. Repeat

9. Plot OD vs time

10. Repeat assay with differen lyzosyme amounts

Is the decrease in OD600 linear or exponential? Suggest two reasons to explain this.

Yes

• rate of reaction is proportional to the amount of intact walls present

BUT

• as attached, disintergretes walls scatter into pieces less light then the original cells

• Fragments could be substrates for lysozyme BUT further digestion would be invisibible→ so does not show rate of lysozome progress!

Is the initial rate of lysis a linear function of the amount of enzyme?

• Must measure it from the linear part of the plot

• Sometimes it is easier to find the log plot but this is not always the case

  • and this finds the first order rate constant!

Variation in the amount of enzyme used

• Doubling the amount of enzyme DOES NOT quite double the rate of hydrolysis

• But the curve is quite straight at lower enzyme concentrations

• Shows an apprent approach towards satuation

Due to:

• difficultires measring the initial rate when rates are very fast

• unsual balance between enorcous substrate and tiny enzyme

  • not the usual case for how enzymes are used??

Activity of lysozme dependso n 2 amino acids

1. Glutamate 35

2. Asparatate 52

How to test effect of pH

1. Use different buffers

2. record control of bacteria at different buffers

3. Plot the initial rate of attenuance drop against pH of the assay

Conclusions

• Optimum pH is 6

• relevant values for these dissociation pKa being 6 and 4.6 for Glu and Asp

Affect of temperature

1. Incubate in sifferent water baths

2. use marble as a stoppper

3. wait for test tube to cool down

4. plot intitial rate of attenuance drop against temperautre

What is the effect of heating on lysozyme activity?

• 80 degrees→ lost half of its activity

• someties precipate formed at hiest tmepratures

If lsozyme was mixed with the assay buffer BEFORE heating

• survived higher tmepraures better

• therfore: stability can depend strongly on pH

• Because enzymes with their cofactors or substrate boud→ more stable and less easily denatured

How is lysozyme so stable?

• small→ less to go wrong

• constian 4 SS bridges

Adding DTT to the enzyme

• broke the SS bridges

• which are needed for heat stability

How to test lysozyeme activity from tears

1. Dilute tears ten fold

2. use as assay as before

How much lysozyme activity did the bodily fluid include?

• Tears have lots of lysozyme activity, equivalent to about 2 mg/ml of the purified enzyme we supplied

• Egg white has about 3 mg/ml lysozyme

• Sweat usually has up to 0.2 mg/ml lysozyme and saliva 0.1 mg/ml lysozyme.

Why does the activity of lysozyme not return upon cooling?→ Test eg white with SDS and/DTT

Results show before and after boiling

DTT + SDS→ DTT reduces the disulphides, SDS reversvres aggregation and gets the precipitated protein back into solution

Once denaturation starts

• makes it much easier to carry on

• A small change in conditions can cause an enormousus increase in rate of denaturation

• Can be due to

  • pH, temperaure, change of solvent

Why is denaturation irreveerible?

Chemical changes taken place

• other amino acids are potentially unstable:

  • Amides→ turn into acids (asparatic etc)

  • Methionine→ oxidised into methionin sulphoxide

SDS

• detergent

• helps protein binding→ gives them strong negative charge

• needed for eggs to stop denaturation

• Also wraps begative charge around→ for SDS polyacrylimide gel electrophoresis

DTT

• reducing agent

• helps to stop deanturation

Afinsen’s experiment

1. Added Urea and DTT upon catlytic activity of ribonucluase

2. Lost all activity

3. Dialysed the Urea out first→ refolded correctly

4. Oxidised the DTT out→ reformed 90% of intitial activity

If oxidised first and then urea out→ only 1-2% of activity

• this is because illegitmatie random disulphide bonds were able to form when the protein was unfolded prior to urea removal!