Experiment 4: Heme extraction from myoglobin protein (~10-20 minutes; Fig. ) Proteins are made of amino acids having hydrophilic and/or hydrophobic properties and form bonds with cofactors. Oxygen-transferring protein myoglobin utilizes a histidine amino acid to bind to the iron center of heme, which is a largely hydrophobic molecule and easily detectable as red in color. We demonstrated the chromogenic property of heme, principles of acid/base chemistry, and methodologies of an organic extraction by extracting the heme from equine heart myoglobin. In this demonstration, heme was separated from myoglobin through the addition of hydrochloric acid. Protonation of hemeligated histidine lysed its coordination bond with iron, releasing heme into the solution. The free heme was then separated from the unfolded protein using the organic solvent, 2-butanone, which is immiscible with water and formed 2 layers when combined with aqueous, acidified myoglobin (Figure ). Because the hydrophobic heme was released from the protein, it moved to the organic butanone layer on top and colored the layer red while the apoprotein remained in the colorless aqueous layer at the bottom. Sodium hydroxide was then added which hydroxylated heme and turned the solution green.