I just had a molecular bio lab in which we compared rates ofenzyme-catalyzed reactions (in this case forming of benzoquinonefrom catechol in potato extract with added catechol) in variedconditions by analyzing absorbance via spectrophotometer. As Iunderstand it, darker solutions (and higher absorbances) wouldindicate a faster reaction. So conditions that deactivate theenzymes (cold, or very low ph) would result in slower reactions /lower absorbance. The actual spectrophotometer results seemincorrect to me, and as the lab was done by a group of 4, I did notparticipate in every step, and am having trouble identifying theerror.
For one part of the experiment we incubated (or iced) samples ofpotato extract with catechol at various temperatures; for anotherpart we added ph buffers to the potato extract/catechol solutions.After 5 minutes, each sample was run through the spectrophotometer.Different blanks were used for calibration for the temp and phruns.
For the temperature experiment, we saw HIGHEST absorption atlowest temp (4 degC) and LOWEST absorption at room temp. Actualabsorbances: 4deg / .469, 25deg / .436, 37deg / .474, 65deg / .493deg
For the pH experiment, we saw HIGHEST absorption at lowest pH,and LOWEST absorption at ph7. Actual absorbances: pH3 / .620, pH5 /.424, pH7 / .291, pH9 / .287, pH11 / .329.
To me these look like the correct trends, but inverted. Whatkind of error might explain this?
a problem with the blanks? an incorrectly calibratedspectrophotometer? some mistake in the creation of the potatoextract? I can vouch for the correctness of the ph buffers, as idid that step and was very very careful about labeling and usingclean pipettes for everything... all the samples were checked forabsorbance at exactly 5 minutes (carefully staggered so 1 was readyevery 2 minutes). all the cuvettes I touched (the pH-buffersamples) were carefully wiped with kimwipes when put intospectrophotometer...
Thanks!
I just had a molecular bio lab in which we compared rates ofenzyme-catalyzed reactions (in this case forming of benzoquinonefrom catechol in potato extract with added catechol) in variedconditions by analyzing absorbance via spectrophotometer. As Iunderstand it, darker solutions (and higher absorbances) wouldindicate a faster reaction. So conditions that deactivate theenzymes (cold, or very low ph) would result in slower reactions /lower absorbance. The actual spectrophotometer results seemincorrect to me, and as the lab was done by a group of 4, I did notparticipate in every step, and am having trouble identifying theerror.
For one part of the experiment we incubated (or iced) samples ofpotato extract with catechol at various temperatures; for anotherpart we added ph buffers to the potato extract/catechol solutions.After 5 minutes, each sample was run through the spectrophotometer.Different blanks were used for calibration for the temp and phruns.
For the temperature experiment, we saw HIGHEST absorption atlowest temp (4 degC) and LOWEST absorption at room temp. Actualabsorbances: 4deg / .469, 25deg / .436, 37deg / .474, 65deg / .493deg
For the pH experiment, we saw HIGHEST absorption at lowest pH,and LOWEST absorption at ph7. Actual absorbances: pH3 / .620, pH5 /.424, pH7 / .291, pH9 / .287, pH11 / .329.
To me these look like the correct trends, but inverted. Whatkind of error might explain this?
a problem with the blanks? an incorrectly calibratedspectrophotometer? some mistake in the creation of the potatoextract? I can vouch for the correctness of the ph buffers, as idid that step and was very very careful about labeling and usingclean pipettes for everything... all the samples were checked forabsorbance at exactly 5 minutes (carefully staggered so 1 was readyevery 2 minutes). all the cuvettes I touched (the pH-buffersamples) were carefully wiped with kimwipes when put intospectrophotometer...
Thanks!
