Biochemistry 125 Sandbox

📚Introduction

Riboflavin is a water-insoluble B vitamin (B₂) which serves as a metabolic source of flavoprotein enzyme cofactors such as flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). Solutions containing riboflavin are colored yellow, and exhibit fluorescence using excitation and emission wavelengths of 280 nm and 525 nm, respectively.

The transparent yellow coloration of some chicken egg whites is a manifestation of the presence of riboflavin. Egg whites contain an excess of the riboflavin binding apoprotein (RfBP) which binds riboflavin molecules with considerable specificity and high affinity. The specific protein-biomolecule interaction actually “quenches” the fluorescent properties of free riboflavin—that is, riboflavin molecules bound by RfBP would cease to fluoresce.

 $Riboflavin+\underbrace{RfBP}_{\textbf{Fluorescent}}\rightleftharpoons \underbrace{Riboflavin-RfBP}_{\textbf{Not Fluorescent}}$

In this activity, riboflavin content of various vitamin samples are quantified using the quenching properties of the riboflavin binding protein. Samples containing riboflavin would fluoresce; upon addition of a diluted egg white titrant, the fluorescence would decrease until a minimum fluorescence is reached (at the equivalence point), corresponding to the fluorescence of the matrix. The difference of the fluorescence after equivalence point from the initial fluorescence (before titration) is directly proportional to the amount of riboflavin present in the sample.

$F_{after\ equivalence\ point}-F_{before\ equivalence\ point}=k\times [riboflavin]$

• Explain the concept behind fluorescence quenching
• Relatively quantify the amount of riboflavin in different vitamin samples
• Explain how protein interactions can be investigated through fluorescence

⚙️Module Tasks

1. Read the protocol for relatively quantitating riboflavin content.
2. Analyze fluorescence data.
3. Write a formal report for this activity. 

⏰Time Allotment: 2 - 3.5 hrs

Suggested Time Frame per Task: