Biology p12          

Physiology 12 Lab , M. J. Malachowski, Ph.D

Enzyme Lab

Introduction

The basic question we are asking is: "Is the activity of the amylase in our saliva temperature dependent?"


If we assume that the activity is not temperature dependent, our hypothesis, then we can test our hypothesis by running an experiment to see if the activity changes as we change the temperature.


Procedure

We will make use of a color indicator to determine the presence of starch. Iodine in aqueous solution is a yellow brown color. In the presence of starch it turns blue black. If we add iodine to a starch solution it will turn blue-black. If we add amylase enzyme to the solution, it will convert the starch to maltose and the solution should turn colorless or yellow brown. If we follow this process over time, we can quantitate the activity of the amylase.


To do this well we need to determine a number of factors. We would like to pick concentrations of starch that amylase enzyme in our saliva will convert to maltose in two to five minutes, at room temperature.


Measuring Color

We will follow the conversion process using a spectrophotometer. Initially, you need to determine the wavelengths you should use to follow this conversion process. To accomplish this, you need to determine the relationship between absorption and wavelengths for your solutions. The easiest way to to this is to construct a plot, graphic representation, of the absorption of the solution in your tubes as you vary the wavelength of the spectrophotometer. See Concentrations

Construct graphs of absorption as a function of wavelengths for iodine-starch and iodine only solutions. Find the peak absorption/transmission wavelengths for an iodine solution and for a starch solution to which you have added iodine as an indicator.


You need to pick concentrations of iodine and starch which allow you to see a change in absorption as you change the wavelength. For this part of the lab, pick a concentration of each solution which has an absorption of less than 100 % (Transmission just greater than zero, i.e., a Value such that when you insert the tube of starch with indicator into the colorimeter, the needle just moves from the left hand side.) at its maximum and just plot the transmission or absorption over the spectral range of the spectrophotometer.


Will you have a problem with the absorption peak of iodine blocking your results of following the change of absorption of the starch-iodine color? Hint: Use one of the tubes that you ran to pick an ideal enzyme amount, which has turned "clear" for your blank. I.e., place this "blank" into the colorimeter and use the right hand know to set the transmission to 100%.


Expected Results

You will determine what concentrations of starch and iodine, indicator, you will use in your experiment. Your first task is to pick values that will allow you to follow the reaction over several orders of magnitude. That is, if your starting concentrations allow 1 % transmission, one order of magnitude would be 10 % transmission and the second would be 100 % transmission. Therefore, you should be able to follow the change over two serial dilutions --- thus, assuming the starting solution is Y = 1/100X (where x was the 1% stock starch solution supplied for the lab), the first dilution is Y/10 and the second is Y/100 x.

For these three concentrations of starch, Y, Y/10 and Y/100, fill three test tubes with 10 ml of each (serial dilutions) For each test tube add indicator, be sure to add the same concentration (amount) of iodine to each tube.

Determine the concentration of each of these solutions based upon the storeroom stock starch solution. E.g. for a 1% starting starch solution - assume a weight of 10 grams/liter. (The % concentration should be on the flask label.)


Enzyme collection and calibration

Each group should collect 25-30 ml of amylase enzyme solution. (You can use parafilm to stimulate saliva production and facilitate enzyme collection.

This can be determined using a variation of the serial dilution. Set up a series of test tubes with your starting Starch-Indicator solution. Pipette an amount of enzyme solution, e.g., 1/10 ml into the first tube, .5 ml into the second, 1 ml into the third, 2 ml. into the third and observe how quickly they become clear. Determine the amount of enzyme solution required to turn 10 ml of starting starch-indicator clear in approximately 2 minutes. Use this value for your experiment.


The result of your serial dilution experiment/paper will be the concentration of your starting Starch solution and the amount of iodine you will be adding to this solution, starting Indicator concentration.


Prepare 100 ml of the Enzyme Lab Starch Stock Solution with the starting Indicator concentration for tomorrow.


(Label your bottle of iodine and save for use tomorrow with the stock starch solution -- if you need more solution tomorrow, you will need to use the same concentration of iodine tomorrow as you used today.)


Next Laboratory Meeting you will run experiments at four temperatures, 0, 20 37, and 50oC. You should run this on the same Spec 20 that you used today. You may need to vary some of the concentrations to obtain results at all four temperatures.


Discussion

Updated 1/25/01 Miko mmalacho@ccsf.org