In this lab we study the activity of enzymes, a very important group of proteins. They speed up nearly all the important biochemical reactions within a cell (which would otherwise take an exceedingly long amount of time) and thus are an indispensable part of life on earth. Specifically, we’re looking at how certain external factors (temperature, pH, etc.) can affect the activity rate of enzymes.
For this activity we’re using the catalytic enzyme amylase, which breaks down starch (a polysaccharide made up of amylose and amylopectin) into maltose (a disaccharide) and dextrin. Humans produce amylase in the salivary glands and pancreas to aid in the digestion of starchy foods, the most common carbohydrate we consume.
How do we use this information to set up a test (assay) for the evidence of enzyme activity, i.e. production of maltose? Well, we know that maltose is a reducing sugar (you all remember redox reactions, OIL RIG and all that, right?) that reacts with and reduces the pale yellow colored alkaline 3,5-dinitrosalicylic acid (DNS) to the orange-red colored, 3-amino, 5-nitrosalicylic acid after being heated for 5 minutes. Essentially, the intensity of the color is proportional to the concentration of maltose present in the solution (per Beer’s Law). Thus, increased amylase activity produces more maltose, which reduces more DNS, which then turns the solution a darker orange-red.
Once we’ve done that, we can use a spectrophotometer to do a colorimetric analysis of samples and figure out how much maltose was produced in different conditions. A higher absorbance will correlate with the darker color (higher optical density). We use a wavelength of 540nm, which is the lambda max for the orange-red color of the “reduced” DNS. The resulting numbers can then be graphed to see the pattern of enzyme activity in changing external conditions.
Introducing the spectrophotometer into the lab is a good way to show students the difference between qualitative and quantitative analysis.
Now that we figured out a test for the presence of maltose, and hence the activity of amylase, what do we use as the substrate? As you know, we use amylopectin (from corn). But why don’t we just use a starch solution with potato or corn starch instead? Remember that starch is made up of amylopectin and amylose, both polysaccharides. Amylose is a linear, helical (more tightly packed) polymer of glucose while amylopectin is a highly branched polymer of glucose. Those branches in amylopectin provide more surface area for the amylase to break it down into maltose units. So, using amylopectin will yield a slightly higher production of maltose during the 10 minutes allotted for the reaction than using either starch or amylose.
Which brings me to the next question, what’s the purpose of the tartrate solution? Remember, maltose is a reducing sugar What do you think will happen if it reacts with dissolved oxygen in the solution? We add the tartrate to prevent oxidation of the maltose and stabilize the color.
Before we get to the lab procedures, let me mention a few things to keep in mind before actually running the lab with your students.
1.- Set up the water bath at least 2 hours before the start of the lab. If it’s the first period of the day, it might be better to set the temperature at about 6 and leave it on over night, then in the morning raise the temperature to the highest setting. Remember, use only distilled water!
2.- Make sure the Brinkmann Dispensettes are pumping correctly and primed before the students use them. There should be no air bubbles in the glass cylinder or discharge tube. You’ll have to go over proper operation with your classes. Point out that the piston must be raised slowly and evenly, then gently pushed down.
3.- It would be very helpful to have students practice using the micropipettes before doing the lab. The accuracy of the data obtained by each group is affected by how well they measure and dispense the different volumes required. So, a little practice beforehand will help tremendously. Have students pay close attention to where the three “stops” are located, they should be able to feel where each one is. Also, make sure they keep track of the upper and lower limits for each micropipette. They should never go below 100uL! They will jam the micropipette if they do this. The 50uL of amylase needed for test tube 2 in Part B will be measured using a different micropipette set aside just for this task.
4.- Timing matters! We’re measuring the activity of an enzyme under different conditions during a set amount of time (10 minutes). It’s important that students understand when the timing should start, i.e. when enzyme and substrate combine. It seems an obvious point, but many students don’t get it at first. They need to be aware of when, for the part they’re working on, they’re mixing the enzyme and substrate and be ready to keep track of the reaction time. Something that can be asked to confirm understanding of the process behind the lab procedures is whether it matters if you add the reagents with the dispensette before adding the reagents with the micropipettes. Of course for Part A and D it doesn’t matter, but it will for Part B and C.
Part A: Measuring the Amount of Maltose Present in a Sample.
The data obtained from this part of the lab will be used to create a standard curve pairing an absorbance reading with known amount of maltose.