Ted M. Fancolli
and Salvatore F. Rurro' Sacramento State College Sacramento, California 95819
Interaction of a Naphthalene Dye
with Apohernoglobin
Hemoglobin is the main component of the red blood cells, and is the compound responsible for oxygen transfer from the lungs to tissues, as well as the transfer of carbon dioxide back to the lungs. Because of its physiological importance, hemoglobin has been extensively studied. This has been facilitated by the ample supply of this material and the ease with which it can be crystallized. Hemoglobin is a conjugated protein of molecular weight 67,000 made up of about 10,000 atoms of hydrogen, carbon, nitrogen, oxygen, and sulfur, plus four atoms of iron. Each iron atom lies at the center of a group of atoms in a porphyrin molecule known as heme. Each heme group is encircled by a chain of amino acids, one of four chains that together comprise the molecule of hemoglobin. The four chains of the protein portion of the molecule are called globin (also called apohemoglobiu). The four chains of globin consist of two parts: the alpha pair and the beta pair. Heme alone will not bind oxygen; yet the specific combination of heme with globin enables the four iron atoms within the molecule to actively take up oxygen. Myoglobin is related to hemoglobin by the fact that it is a quarter of its size. It consists of a single polypeptide chain of about 150 amino acid units together with a single heme group. The sequence of myoglobin is related to the sequence of the a chain of hemoglobin suggesting that these proteins have a common ancestor (1). Myoglobin is contained within the cells of the tissues, and it acts as a temporary storehouse for the oxygen brought by the hemoglobin in the blood. The three-dimensional structure of myoglobin was solved by Kendrew and coworkers ( 2 ) ;it was the first protein to be analyzed by the technique of X-ray crystallography. The exact tertiary structure of horse oxyhemoglobin in the solid phase has been established to a resolution of 2.8 A by Perutz and co-workers (3). As in the case of myoglobin, it has been found that the heme group is in the interior of the molecule, surrounded almost entirely by nonpolar residues. The other part of this narrative has to do with the fluorescence of small compounds (4, 5) and how this relates to the structure and function of protein molecules. I n 1954 Weber and Laurence (6) reported a class of compounds that has the interesting property of being non-fluorescent in aqueous solution but fluorescent when bound to protein. One example is l-anilinonaphthalene-8-sulfonate. It was noted at that time that solutions of these compounds alone in certain organic solvents are fluorescent. Also, aqueous solutions of 1 Present Address: Chemistry Department, Western Washington %ate College, Bellingham, Washington 98226.
54 / Journal of Chemical Educntion
t h e compounds become brightly fluorescent following the addition of adsorbing proteins such as serum albumin or heat-denatured proteins. There is good evidence that ANS binds to hydrophobic sites on proteins. Hydrophobic bonding is one type of noncovalent force which is important to the maintenance of tertiary structure in proteins. Thistermwas first used by Kauzmann (7) to describe the tendency of nonpolar residues to avoid the aqueous phase and adhere to one another. Another viewpoint concerning the nature of hydrophobic interactions has been expressed by Iilotz (8). For a recent review of this important aspect of protein structure see the article by NBmethy (9). Stryer (10) has studied the binding of ANS to apomyoglobin and apohemoglobin. He found that one could insert one molecule of ANS into apomyoglobiu and one molecule of ANS per apohemoglobin quarter molecule. The fluorescence quantum yield of ANS is essentially zero but in ANS-apomyoglobin it reaches a value of 0.98 and in ANS-apohemoglobin i t is 0.92, both of these values being close to the theoretical limit of 1. The ANS in the fluorescent ANS-apomyoglobin complex could be expelled by adding hemin which is consistent with ANS and heme being bound to the same site. One should recall a t this point that Iiendrew and Perutz have unequivocally established that the heme binding site is in the interior of myoglobin and hemoglobin and is surrounded almost entirely by nonpolar residues. The ex~erimentdescribed in this communication deals with the noncovalent interaction of ANS with apohemoglohiu. This involved the preparation of hemoglobin and the dissociation into heme and globin. ANS was then added to apohemoglobin to form a highly fluorescent complex. Finally, the ANS was expelled from ANS-apohemoglobin in an experiment analogous to the one performed by Stryer on ANS-apomyoglobin. This experiment is quite challenging on the undergraduate level or it would he suitable for a graduate level laboratory. I t is quite instructive in that it illustrates the preparation of pure protein, dissociation into its constituent parts, and subsequent physiochemical studies.
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