Turnip Peroxidase Reactions

Tips for Lab Reports

Plagiarism – future assignments will receive a “0” ; Dr. Moore will reserve the right to extend/deny a re-write of the lab Changing/rearranging a few words does NOT

sufficiently make it your work If you can’t think of a way to re-write an idea(s) you

must cite the work:Adapted from “Enzyme Investigations for Introductory Courses” by Ruthanne B. Pitkin. in Tested studies for laboratory teaching, Volume 13 (C. A. Goldman, Editor). Proceedings of the 13th Workshop/Conference of the Association for Biology Laboratory Education (ABLE) 1992

How to cite your labs: (Blatant laziness will be penalized) Adapted from Lab 4 “Peroxidase Activity” by Dr. Susan Moore, 2010

Tips for Lab Reports

Hypothesis – Much easier to write as an “If…Then” statement Need enough detail to actually say what

you’re testing and that it can actually be tested

Can only be accepted/rejected, NOT proven true, inconclusive data = reject

Wrong: I hypothesize that caffeine with raise Daphnia heart rate.

Right: If Daphnia are exposed to increasing concentrations of caffeine, then we hypothesize that the heart rate will increase proportionally.

Tips for Lab Reports

Methods section – Most importantly avoid plagiarism Put it in your own words with ENOUGH

detail that the experiment could be repeated

Should always mention what software was used to calculate statistics and generate graphs

Example:Microsoft Excel was used to calculate all descriptive statistics and in the generation of the column graph.

Tips for Lab Reports

Results section – should contain statements about the data found and is a space to enter charts and graphs NOT an area to talk about what can be concluded

Example of “results – type” statements:Daphnia were exposed to a series of eight, 10-fold dilutions. The highest concentration, 20.0 mg/L, showed the highest heart rate of 320 bpm.

Enzymes

Enzymes are essential for cell function and catalytic reactions.

Most enzymes are proteins, with certain RNA molecules functioning as enzymes as well.

Enzymes are regulated/affected by numerous things, including inhibitors, pH changes, temperature, etc.

EnzymesA substrate binds to

an enzyme Enzymes are selective

for specific substratesCan speed up

chemical reactionsThey release end

products which are different than the substrate

Enzymes

The theory and functioning of enzymes will be studied over the course of the next two labs.

In this lab, we will look at the activity of the peroxidase enzyme, acquired from a turnip, and how different concentrations of the enzyme affects reaction rates.

Peroxidases donate electrons to bind to other substrates to break them down into harmless products

peroxidaseRH2 + H2O2 → R + 2H2O

2 H2O2→ 2 H2O + O2

The reaction serves to convert toxic hydrogen peroxide (H2O2), a product of metabolism, into water and oxygen. This reaction occurs spontaneously, but slowly, in the absence of peroxidase.

Tetraguaiagol

2

How can we follow this reaction?

If we add more enzyme (turnip prep), will the reaction go faster?

What are the optimal conditions for this enzyme?

Materials/Procedure

Work as group of 4.Bring back stock solutions to your table

( label 3 tubes). Fill two small test tubes (half full) of 0.1% H2O2, guaiacol and 1 full test tube of turnip enzyme prep(TEP). Place all supplies on labeled paper towel

BE ORGANIZED!Label 6 test tubes and follow the direction in

your lab manual for the amount.Use parafilm to invert and mix the substrates

in your test tube. BE GENTLE.

Pipetting and Pipet Pump

We will again be using Fisherbrand Pipette Pumps

Before you begin measuring out the substrate you should practice with the pipettes labeled “practice” and the colored liquids.

Do not invert your pipette/pipette pump!Place used pipettes on labeled paper towel

so each is only used for designated solutionOnce completed used pipettes will go into

specified boxes so they can be reused for next weeks lab

Reminders

Make sure to use clean pipettes for each of the solutions you are working with. DO NOT MIX AND MATCH! Contamination with different liquids will skew your results.

Use the proper pipettes for the proper amounts of liquid. Make sure to adjust your pipette pump appropriately when switching from 10mL to 5mL, etc.

Reminders

Label your tubes and pipettes as needed. Use paper towels to write on.

Wear your safety glasses and gloves. Guaiacol is an irritant and harmful to your skin and eyes.

Materials/Procedure Cont

You will be using a spectrophotometer to analyze the rate of the peroxidase reaction. This device quantifies the change in color brought about by the reaction.

From Blackboard tutorial, you should be knowledgeable in how to use this device.

Follow the lab protocol for zeroing and using your particular spec

SpectrophotometerNeeds to warm up for

about 15 minutesWavelength should be

set to 500 nmFirst need to zero

instrument (zero absorbance)

You will test each sample solution at the specified time and read the absorbance value

VERY IMPORTANT!!

You must read the whole procedure before you begin!!

The reaction will begin occurring as soon as you add the enzyme, this is a TIMED experiment so make sure you have everything organized and ready!

AKA – Get all tubes set up as in Table 1 of the protocol before you proceed with adding the turnip enzyme

Clean-Up

Remember to rinse and clean your test tubes. Leaving a mess and not cleaning up will result in loss of performance points!

Organize materials into green pans as they were found.

Used pipettes and other materials – place into boxes as indicated.

Wipe and dry desk

Next Weeks Lab & Homework

You will plan an experiment for next weeks labYour group will pick a variable to experiment –

next slidePrelab 5 Assignment will be due at the

beginning of lab next weekEven though you will do the experiment in

groups of 4, all work on the assignment needs to be individual (DO NOT COPY!)

The assignment also includes some new graphing

Choose Your Variable

Temperature – see water baths for your options

pH – see the display as to which pHs will be available

Salt – see the bench for dilutionsEthanol – see the bench for dilutionsDetergents – see what’s availableMechanical disruption – through the use of

small bore pipetsSubstrate concentration – alter the

concentration of the substrate for the experiment

Graphing

There will be detailed instructions for adding trendlines and fulfilling the assignment requirements on BlackBoard

See BlackBoard for the posting of the assignment and Dr. Moore’s document on help with graphing and laying out data

If you have any problems email us or come to our office hours

Before You Leave!Look around the room

at the available variables, get specific information for your groups variable (exact temps available, detergents, or conc.)

Pick up your graded Prelab 2 assignment and Lab report

We will check your notebooks before you leave!