Bio Lab 7 - Enzymes II

milk concentration of protein

1g/fl oz. (1fl oz. = 30mL)

What is the major milk protein called

casein

How does protein dissolve in water

must be surrounded by hydrogen shells (coats of water). Hydrogen of the molecules reduces their interactions so they don’t stick together and form precipitate.

Altering 3-D structure of protein

• casein is precipitated

• Happens when contaminating bacteria in milk produce lactic acid as a byproduct of metabolism

What happens when pH of milk decreases

casein becomes less soluble and precipitates as casein molecules interact with each other more than the water molecules. This is seen as curdling and the “spoilage” is used to make cheese and yogurt products. The precipitated casein can also be separated as curds leaving the liquid part as whey.

Effects of pH experiment

dispense 3mL of 4M HCl into a plastic tube then add 25mL of nonfat milk and mix. Curdling should appear (casein precipitate)

Effect of organic solvent experiment

9mL of milk in a plastic tube than add 19mL of 95% (v/v) ethyl alcohol. mix and precipitate will appear

Centrifugation experiment

if liquid has solid within then the solid and liquid can be separated using centrifugal force. The greater the force the smaller the particles that can be forced to the bottom of the tube

What are the separated materials when centrifuging

The casein “pellets” move to the bottom (solid)

the liquid “supernatant” moves to the top (contains lactose, minerals, and vitamins)

Why is one pellet smaller than the other?

The tube with HCl which dropped the pH separated the caused the milk to become insoluble by separating. the casein and causing clumps

The organic solvent, ethyl alcohol added disrupts hydrogen shells and effects solubility but does not separate as thoroughly.

Partial purification

• Pour the supertanant in the sink but leave the pellets

• Put 15mL of water into the tube with pellets

• A new solution containing the proteins but none of the other solutes is formed

Concentrating protein

The new solution only containing the protein found in milk and water is only 15mL of added water rather than 25mL of the original volume (important use of centrifugation)

What happens to the proteins exposed to acid and alcohol

In both samples the proteins denatured

Effects of acid

• H+ protons are added to amino side chains that disrupt ionic bonds and salt bridges that hold protein structures together

• Loss of repulsion between casein molecules at low pH causing clumping

• Hydrogen shells are disrupted and hydrophobic regions are exposed causing protein aggregation and precipitation

Effect of ethanol

• Alcohol interferes with secondary and tertiary structures of proteins

• Alcohol competes with the water molecules attached to proteins and removes hydration shells (decrease solubility)

• Hydrophobic amino acid regions are exposed and clump

  Casein precipitates slower and less fully with ethanol rather than the acid

Calibration of colorimeter

• Plug in colorimeter and allow to heat for 5 minutes

• Have blank prepared (accounts for glass, water, and both substrates Guaiacol and H2O2)

• Click %Transmittance vs Time

• insert blank until finish calibration pops up

• Set wavelength to 500nm and click done

Assay procedure

• Put 0.2mL of enzyme in large test tube

• Put all other solutions in second test tube

• Pour second test tube into tube with enzyme (reaction begins immediately and color forms right away)

• Immediately pour into cuvette and place in colorimeter

• measure time it take for %T to fall from 70% to 50%

• calculate reaction rate (200 micromoles/Time from 70% to 50%)

Enzyme activity and pH

Every enzyme functions best at a certain pH or pH range. Above or below that range the enzymes activity is less

pH effecting charge

pH change can effect charge of amino and carboxyl groups in amino acid side chains changing charge-charge interactions and altering 3-D structure. Enough pH change can denature the enzyme

Preparation of dilution series of HCl

• set up test tubes labeled 2-7

• pipette 4.5mL of water into each tube

• In tube 7 pipette 0.5mL of 2.5×10^-1M HCl (this dilutes by 1:10 making a 2.5×10^-2M HCl solution)

• mix

• Continue this with other solutions with continuous dilution factor of 1:10 by pipetting 0.5 of the last tube into the next (ex. 0.5 2.5×10^-2M HCl from tube #7 goes into tube #6 to make 2.5×10^-3M HCl)

How to calculate dilution factor

initial volume/final volume

ex. with HCl for tube 1

0.5/(4.5+0.5) = 1/10

contents of assays in which pH varies

• Each assay has 5mL total

• H2O2 and guaiacol substrate vol. are constant

• enzyme vol. is constant

• 2mL of differing HCl conc. are added to solution

• Another dilution of HCl occurs again

How to do second dilution, calculating final HCl conc. (ex. using #2)

• dilution factor: 2mL HCl/5mL total = 2/5

• New conc (initial x dilution factor): 2.5×10^-7M x 2/5 = 10^-7M HCl

Calculating pH

pH = -log[H+]

ex. (#2)

pH = -log[10^-7] = 7

What to do after calculations

use a 2mL pipette to measure HCl solutions and perform assays recording time from 70% to 50%

results from different assays

As assay # went up so did concentration of HCl. Higher conc. of strong acid caused reaction rate to decrease and a longer time to go from 70% to 50%

Acidic environment eventually denatured enzyme and destroyed active sites

Why was did assay #7 yield its results

drastically lower reaction rate because protonation of key amino acids at active sites disrupted substrate interactions and decreased solubility

Hypothesis: What if enzyme did not denature

• High H+ conc blocked reactions in another way

• If pH did damage the enzyme then even when returned to a suitable pH environment the enzyme still should not work

• If added base causes enzyme activity to begin than the enzyme was not denatured

What does assay #8 test for

If the enzyme was denatured by acid or if the H+ block the rxns in a different way

Assay #8 set up

• One tube with 2mL guaiacol, 0.8mL H2O2, 1.6mL water

• Second tube with 0.2mL enzyme and 0.2mL of 2.5×10^-2M HCl

• After 30 seconds add 0.2mL of 2.5×10^-2M KOH (should neutralize solution)

• After 30 seconds pour first tube into enzyme tube

• record time

Assay #8 results

short time to go from 70% to 50% so hypothesis was supported

What does assay #9 test for

effect of high temp on enzyme activity

assay #9 set up

• Pipette 0.2mL enzyme into a test tube and leave in hot water for 60 seconds

• cool the tube to room temp

• combine other solution as usual (2.0mL guaiacol, 0.8mL H2O2, and 1.6mL water)

• measure time from 70% to 50%

Assay #9 results

• very slow reaction rate or does not drop from 70% to 50%

• High temp caused enzyme to denature

• IMF’s were broken

• Active sites were destroyed

• protein structure was altered

Assay #10 purpose

Show effect of organic solvent on enzyme activity

Assay #10 set up

• combine 0.2mL enzyme with 1.0mL ethanol in test tube

• Combine other solutions as usual (2.0mL guaiacol, 0.8mL H2O2, water)

• Time from 70% to 50%

Assay #10 results

• Organic solvent denatures protein

• IMFs are disrupted

• Enzyme unfolds and aggregates

• Protein solubility decreases when nonpolar regions are exposed

Assay #11-13 purpose

show effect of an irreversible metabolic inhibitor on enzyme activity

Irreversible inhibitor

inhibitory effects cannot be undone or reversed. Inhibitor molecule attaches to enzyme and stays there permanently shutting down enzyme. Does not denature or destroy folded structure.

NaF inhibiting peroxidase example

NaF enters peroxidase active site and permanently binds so the substrate can never enter the enzyme

assays #11-13 set up

• combine enzyme and NaF in one test tube

• Prepare another test tube with usual contents except guaiacol is 0.1mL rather than 2mL

Results of each assay 11, 12, and 13

• 11 is a control with no NaF: Rxn proceeds normally

• 12 shows effect of NaF on rxn rate: Rxn stops, NaF blocks active site

• 13 tests if NaF can be undone by increasing substrate conc: Rxn stops even with increased guaiacol conc. showing inhibitor is irreversible

Reversible inhibitor

inhibitory effects on enzyme can be reversed (in this lab its catechol). Does not permanently bond to active site

Catechol

reversible and competitive inhibitor with very similar structure to guaiacol. -OH group is in place of -OCH3. Similar structure allows it to enter active site but difference in functional group does not allow rxn to proceed. Catechol and guaiacol will compete for active site.

assays #14-17 set up

• pipette enzyme in a large test tube

• put the usual concentrations (lower guaiacol) in a separate tube

• mix

Assay results

• Assay 14 was a control: no catechol so no competition and normal activity

• Assay 15 showed catechol effect: rxn rate drops

• Assay 16 double the guaiacol conc was present: better chance of substrate reaching active site so rxn rate improves

• Assay 17 no substrate: no rxn showing catechol is not a substrate and an inhibitor