Fluorometric Analysis of Riboflavin An Undergraduate Biochemistry Experiment Ji-Yue Hu and Edward G. Senkbeil' Salisbury State University, Salisbury, MD 21801 Harold B. White, Ill University of Delaware, Newark, DE 19716 Fluorometry is an important analytical method that demonstrates a high degree of both sensitivity and selectivity for manv comoounds. There are few published undergraduate expekmen& that adequately demonstrate this technique ( I ) . Especially in biochemistry, the fluorescence quenching due to specific binding of a ligand by a protein is an important analytical tool. In this experiment, the fluorescence of rihoflavin (vitamin Bp) is specifically quenched when titrated with riboflavin-binding protein (RfBP). From such a fluorescence titration, the amount of riboflavin in an unknown solution and the dissociation constant of the vitamin-protein complex can be determined. The experiment can be completed in a 3-h lab period with some advance preparation by the instructor. Riboflavin, a n intensely fluorescent compound (21, is required by all higher organisms. I t is a precursor of two closely related coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which function as prosthetic groups of flavoprotein dehydrogenases. The fluorescence of riboflavin is quenched upon binding to a specific riboflavinbinding protein found in yolk and white of bird eggs (3). Riboflavin-binding protein transports riboflavin into the eee (.4 ) where the stoichiometric vitamin-orotein comnlex contains 12.5-12.9 ,tg of rihuflavin bound per mg of R ~ B P . For this exneriment. the riboflavin-binding nrotein need not be purified. ath her; rihoflavin is simply removed from the complex in egg whites (below). The general method has heen utilized for urinary analysis of riboflavin (5) and has also been recently presented a s a qualitative biochemical demonstration of the formation of a vitamin-protein complex (6). ~ m , .
Prlnclple of the Method Riboflavin-binding protein hinds specifically with riboflavin (Rf) in a 1to 1ratio and quenches its fluorescence,
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RfBP Rf = RfBP .Rf fluorescent not fluorescent
(1)
A riboflavin sample is excited a t 447 nm, and its emission fluorescence is measured a t 530 nm. The assay sample (5.0 mL) should be maintained a t p H 6 8 for fluorescence stability. T h e riboflavin-binding protein solution is added to the assay ruhe in 10-#L increments until no rhange in fluorescence iu observed. The fluorescence should not require more than 100 UL of HfUP solution such that dilution is not a factor. he riboflavin is quantitated utilizing the change in fluorescence and a rihoflavin standard curve.
Chemicals and Equipment Chicken egg Buffer: 0.05M Tris-HC1pH 7.5
Dialysis tubing (maximum molecular weight cut-off of 20,000) Riboflavin HCl Preperative centrifuge Fluarometer (Sequoia-Turner Model 112 or equivalent) Preparation of Riboflavin-Binding Protein Break an egg and separate the white fmm the yolk. Theegg white is diluted with an equal volume of water, stirred magnetically for 10 min to disperse the gelatinous egg white, and then centrifuged for 10 min at 7700 X g. The supernatant is dialyzed (with stirring) for 24 h against four changes of 20 volumes of 6 mM HCI. This method of preparation of the riboflavin-bindingapoprotein is based upon the observation that rihoflavin (which is found naturally hound to the protein in eggs) dissociates fram the protein at low pH (7). The sample is then dialyzed (with stirring) for 24 h against 20 volumes of 0.05M Tris-HCI pH 7.5 and centrifuged. The crude rihoflavin-binding protein solution from one egg white is adequate for about 500 riboflavin assays. It may he frozen in l-mL aliquots for later use. Commonly, a 5 to 1 dilution of this solution is used for the actual riboflavin assay. An alternative to this procedure is to purchase the apoprotein from Sigma Chemical Company (Cat. No. R-8628). Preparation of Riboflavin Standard Curve A stock riboflavin solution is prepared by dissolving 18.0 mg of riboflavin in 500 mLof distilled water containing 1 mL concentrated HCI. This stock solution isstable for 2 months at 4 T , provided it is kept in the dark. The riboflavin stack solution is diluted 1:200 with distilled water to oreoare a workine standard. Aliauots (0.2.0.4.0.6. O H . 1.0 mL) of rhis.norking srnndard are dilutid to 10 mi. with 0.u5M Tns-HCI pfl 7.5 for rhe ril,utla\,in standard i:urre suluriuns. Thr calibralien plot irihaflsvin cmrenrration vs. fluorrscenct units1 is linear o w r the rnnke usrd, producing n correhlion radiirim1 of 0.998 and B sensitivil? t81 r,f 4 25 fluoreicenre imn, ng riboflavinpermL.Ths h i t ofdetection 16)ii0:ll ngrihdlsvtn m l . at the 95%eanfidence level Analysis of Unknown Riboflavin Solution Arihoflavinsolution of unknown concentration (in the range of 520 ng/mL) is prepared and its fluorescencerecorded. 10-pLaliquots of the rihoflavin-bindine ".orotein solution are added until the fluoreseenee no longer decreases. Estimation of the Dissociation Constant for Riboflavin Using the rihoflavin standard curve and the titration from the preceding section, students will be able to determine the concentration of riboflavin in the unknown solution. This concentration can then be used to analyze the titration curve and estimate a dissociation constant (Kd) for riboflavin. Using a ruler, extend the line between the first two points (Fig. 1)to the x axis. The intercept correspondsto the volume of egg white containing one equivalent of riboflavin-bindingprotein. The axes of the titration curve can now be converted from fluorescence units and volume to the concentration of riboflavin IRf) and total rihoflavin-binding protein, [RfBP] [RfBP.Rf]. The concentration of protein-bound rihoflavin, [RfBP. Rfl, at any point on the titration curve can be estimated fram the difference between it and the initial point. Students should select several experimental points near the equivalence paint and calculate the dissociation constant (eq 2) for each.
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' Author to whom correspondence should be addressed. Volume 67
Number 9
September 1990
803
