More about proteins and lab technique

if a proteins structure is disrupted what happens

changes shape, denaturation, loss of function, percipitates

what helps keep a protein from clumping

hydration shells

If an acid is added to protein what happens

pH decreases

solubility lowers and changes shape of proteins

protein stops interacting with water and interacts with itself

if alcohol is added (ethyl) what happens

similar to acid, denatures protein

protein clumps to “fluffy percipitate”

What does centerfugation do, explain process

spinning tube: separate solid and liquid

• pellets on bottom

• supernatant lquid on top

what is pH

amount of H+ in solution

What can pH do to structure and how does it do this

denature enzyme

creating charges on amino and carboxyl groups to destabilize structure

why do we cosat the pipette with new solution and why can we use the same pipette

• coating with concentration thar is correct

• bc its the same solution just diff amount

To determine pH calculation, what are the equations used

Use Assay #2 as example

C1V1=C2V2

(2.6×10^-7)(2)=C2(5)

C2=1.0×10^-7

pH= -log (10^-7)

pH=7

If you lower pH, what should happen to the reaction rate

Reaction rate decreases

What was the hypothesis we tested with assay 7?

enzyme is not denatured by low pH, but high H+ is just blocking the reaction in some way

when testing the hypothesis what can we look for?

if it is denatured, fixing pH wont do anything

If it is not denatured, neutralizing acid will help acid work again

What do we use to neutralize and test the hypothesis? What do we use it on?

KOH on HCl

What was the outcome of the hypothesis?

Reaction is still very long and pH is likely denatured

What are the effects of high temperature on enzyme

slows reaction, bc enzyme is more active in cold

what are the effects of an irreversible metabolic inhibitor on enzyme activity

provide example

binds to the enzyme permanently

NaF is inversible inhibitor of peroxidase

how is NaF and Catechol similar and different

both inhibitors

NaF

• irreversible

• does not compete so adding more substrates wont change anything

Catechol

• reversible

• competes with substrate

• depends on concentration

what are the two inhibitors

NaF and Catechol

How does catechol and NaF structure relate to Guaiacol

Catechol - similar structure

NaF - different structure completely

If NaF is added what happens to reaction

slows

if NaF is added with Guaiacol what happens to reaction

nothing changes, it is still slow

if Catechol is added what happens to reaction

it slows reaction, but not completely because it will eventually degrade

if Catechol is added with doubled Guaiacol what happens to reaction

it will happen a bit faster than if Guaiacol was not added because it can now compete with catechol to attach to enzyme

if Catechol is added with NO Guaiacol what happens to reaction

No reaction will occur because there is no Guaiacol to compete