Lab Shedule
Lab 01
Lab 02
Lab 03
Lab 04
Lab 05
Lab 06
Lab 07

Cell cycle game

BIO 196 Official Website

Spectrophotometer & Enzymes

Exercise A (spectrophotometer)

The beginning of the lab spends some time describing the various aspects of the spectrophotometer and the relationship between absorbance and transmittance. The formulas and range of colors are important (how they correspond to the wavelength). Results don't begin until the next section.

Exercise B

Here's where we start observing some trends and results from the spectrophotometer. The first table is 4B-1 and it should look something like this:

Note that you can clearly see the inverse log relationship from this graph. As absorbance increases, transmittance decreases, and vice versa.

Exercise C

Exercise C used varying concentrations of BSA (Bovine Serum Albumin) to test the spectrophotometer's absorbance. The logic is that if you know the absorbance values for varying concentrations, you can use the information to determine the concentration of an unknown by reading its absorbance in the spec. Since every group's unknown was different, I'll just show you what the table and graph should look like for 4C-1.

Note that as the concentration increases, the absorbance increases.

Exercise A (enzymes)

Part 1 of Exercise A investigates the effect of varying temperature on enzyme activity. It's important to remember that the increased absorbance means greater amount of product and a higher reaction rate . Table 10A-1 is a summary of the investigation results:

Remember that all absorbance readings are done at 420 nm.

The greatest activity vs time is displayed from the enzyme at 24 degrees Celsius. At 50 degrees the enzyme is most likely denatured and at 10 degrees it will work for a short time but at a much lower activity as compared to 24 degrees.

Part 2 further investigates the varying effects of external forces on enzyme activity. This time we examine pH. The results are listed below:

The results show that the best pH for our enzyme is somewhere around 6. At other concentrations of hydrogen ions it will not function properly.

The graph for Part 3 gives away a bit of the answer to the next question: how does enzyme concentration effect the reaction? The results are tabulated and graphed below:

Tube A contains no enzyme so the rate is very slow. Tubes C & D have the largest amount of enzymes so the appear to generate the greatest absorbance in the shortest amount of time.

The last section shows how increasing the substrate concentration effects the enzyme's activity.

Yuck! Look at all those tubes! Don't worry, the graph is easier to decipher than you might think. The concentration of substrate increases as the tube number increases (so tube 8 has more than 7, which has more than 6, etc.). As you can see, Tube 8 has the greatest absorbance (= more rxn) in 6 minute time window so therefore the reaction rate is the fastest (and greatest). As you use less substrate, the rate is less and slower. There is an upper limit that will be reached when adding more substrate - eventually you will be limited by the amount of enzyme that is catalyzing the reaction. Therefore, the curves cannot continue to climb higher and higher indefinitely.