Question

In order to measure the rate of this reaction, we utilized the fact that starch - in the presence of iodine has a distinct dark blue color while the conversion of starch to its monomer leads to a more transparent/amber colored solution. While you used a simple visual analysis to verify the conversion, it is also possible to quantify the change in (starch) using a spectrophotometer. 1. Why/how was a spectrophotometer used for this purpose? EXPLAIN what this machine does and how, consequently, it could be used in an enzyme lab. Compare and contrast the concepts of ABSORBANCE and TRANSMITTANCE. 2. You can measure absorbance of the starch solution to indicate the presence/or absence of starch. Will a solution RICH in starch have a higher or lower absorbance value? WHY? Could we have measured transmittance? What would the values have looked like? 3. Given the following information (absorbance values; highest value = 2.0), which solution has the greatest amount of starch? EXPLAIN your answer. a. 0.23 b. 0.89 c. 1.2 4. The following data are the absorbance values for three different concentrations of amylase (values taken every five minutes for twenty minutes). Which data set has been exposed to the HIGHEST enzyme concentration? Which is the LOWEST? Explain your answer. a. 1.0, 0.9, 0.77, 0.69 b. 1.0, 0.7, 0.5, 0.29 c. 1.2, 0.67, 0.33, 0.18 5. Using data set "B" above, what would/could happen to these data values if this SAME experiment were run at body temperature (37 degrees centigrade) instead of room temperature (25°C)? What if we cooled the system down 10 degrees below room temperature? EXPLAIN.

Answer 1

Hi there, as per my guidelines, i would like to answer your first four sub-parts:

1. Spectrophotometer is a machine used to measure amounts of visible light by coloured solutions at specific wavelengths in visible light range. It is used in quantification assays where a desired compound is quantified by complexing it with a coloured compound which imparts it a colour and this coloured compound absorbs light at specific wavelength. According to Beer-lambert's law the amount of light absorbed is proportional to amount of compound present.

Absorbance and transmittance are two important terms. If a compound absorbs light completely then the absorbance is 100% and transmittance is zero i.e. the light left unabsorbed and vice versa.

2. The absorbance value of solution rich in starch will be high because the coloured compound absorbs all the light whereas its transmittance will be zero.

3. solution c. has the highest amount of starch because its absorbance value i.e. the light that the iodine-starch complex might have absorbed has the highest value.

4. solution a. has highest enzyme concentration and c. has lowest enzyme concentration, enzyme amylase is representative of amount of starch present that can be hydrolysed, so higher the amount of enzyme higher is the activity. Set a. has high absorbance values and hence a higher enzyme concentration whereas set c. has comparatively lower absorbance values so the enzyme concentration will be low.

Hope this helps.