Fluorescence; The Crazy World of Wavelengths Week 3

My name is Colton and I’m visiting the Buccella Laboratory at New York University. The lab is part of the chemistry department and we are studying fluorescent compounds that bind with magnesium.

 This week went by pretty fast and I’m already sad that I only have 3 weeks left. Like usual I was up at 4:30am to catch the train and be in the lab by 9:30am. As soon as Sarina came in we began that day’s experiment; testing changes in spectroscopic properties of a compound in different solvents. You may be looking at your screen asking yourself “Well what does all that mean?”. Spectroscopy is the study of how molecules interact with radiation (light). We are testing the fluorescent properties of a molecule. Essentially how much and what type of light a molecule emits and how that changes according to the solvent it is in.  

First, we had to make our solutions using the compound 1C.

Compound 1C
(SCS_2_185 & CK_1_06)

Using DMSO (Dimethyl Sulfoxide) we diluted 10uL(micro-liter) of 1mM (milli-molar) compound 1C to a 2uM (micro-molar) solution. We did the same but in ACN (Acetonitrile). We then began our tests on the UV-Vis and fluorometer. Using the UV-Vis we were able to gather the absorption maximum of each solution. Absorption maximum is the wavelength with the greatest absorbance for a compound. That maximum is then used as the excitation wavelength to gather emission data on the fluorimeter. Basically, that wavelength of light is used to excite the molecule. The emission scan tells us the wavelength that is emitted with the greatest intensity when excited with that given wavelength. The emission maximum (the wavelength emitted with the greatest intensity for a given excitation wavelength), is used in the excitation scan. The excitation scan tells us what wavelength is necessary to have a compound emit a certain wavelength (essentially confirming the absorbance scan). It was unbelievable to see how the fluorescence intensity changed so much in different solvents. Not only did it decrease but it either shifted left or right on the light spectrum. Left shifts move towards blue light, and right shifts move towards red light.  Here’s a graph to give you a visual of the data we collect.

Absorbance Data of Compound 1C in Various Solvents. Peaks represent absrobance maximum.
(CK_1_06/16)

Fluorescence Data of Compound 1C in Various Solvents. 
(CK_1_06/16)

Monday was a long day and we didn't finish up until about 7pm. We were tasked that night to plan for an unplanned experiment to start the next day. So for the time being my experiment was put on hold, because we needed the instruments for this new experiment. The previous week I mentioned that Brismar and I were testing a compound, and as it turned out, we needed to check the compounds binding ability with magnesium in the presence of ATP. The way the compound was designed it only need to bind at two places with magnesium and magnesium has four binding sites. This may seem insignificant, but it’s not because this means magnesium can still bind with ATP, which is not beneficial to our research. The goal of the fluorescent sensors the grad students and Dr. Buccella are designing are to locate free magnesium in the cell, not magnesium bound to other compounds and proteins. So for the rest of the week, that would be the project that Sarina, Brismar and I would be working on.

Before testing anything, we had to make solution of ATP and Magnesium-ATP. It was much more challenging than we were expecting. The original concentration of ATP we wanted in each solution would require more ATP than we had. When we figured out the most concentrated solution we could make with the ATP we had, we didn't account for the Kd (dissociation constant) of ATP, which would not allow us to make the concentration we wanted to, because it wouldn't dissolve in such a little amount of solvent. After an hour or two of thinking, plus doing various calculations we found a concentration that worked. So Tuesday was spent making our solutions. We were finally able to begin the experiment. The first test we did was with the Magnesium-ATP solution. As we preceded with the emission scans, we began to see a decrease in fluorescent intensity as we added more Magnesium-ATP to a solution of the sensor. This was really odd. We figured the ATP binding with magnesium may quench the fluorescence a bit, but not decrease as we add more. Well we did some thinking with Dr. Buccella and we discovered that the equilibrium of the reaction was slow. So when we proceeded with the second trial, we allowed the reaction to reach equilibrium after each addition of Magnesium-ATP. It turns out this was all we needed to do, and instead of decreasing, fluorescence increased, but not as much as when ATP is not present, which means ATP is affecting the fluorescence. (Always do experiments twice… you never know what could happen.)  The rest of the week consisted of us repeating this experiment and doing the same experiment with just ATP, to see how the sensor is affected by the ATP, with without the presence of Magnesium.

To sum the week up, it was very exciting with many surprises. For me one of the best parts was seeing and helping figure out why we were getting the results we got and how we could test them further. It was a lot of critical thinking, but that’s never a bad thing!