This laboratory experiment uses the binding of riboflavin- binding protein (RP) to neutral red (NR) to determine Kd.NR has been used in laboratory experiments to determine an acid dissociation constant using absorption spectrophotometry (8) and mathematical deconvolution techniques (9), whereas RP has been used for determining the quantity of riboflavin with spectrofluorometric techniques (10). The experiment presented here was used with first-year graduate students. On the basis of the class evaluation, the experiment is simple and efficient and can be finished within a 2.5 h period. Although RP can be purchased from a commercial source, students can purify RP
In the Laboratory
from chicken egg white in reasonable quantity using a simple protocol (see the supporting information).