Hemoglobin. Isolation and chemical properties - ACS Publications

alpha chains and two identical beta chains. Heme alone will not bind oxygen; yet the specific combination of heme with globin enables the 4 iron(II) i...
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Salvatore F. Russo and R O I P ~B. Sorstokke Western Washington Stote College Bellinghom, 98225

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Hemoglobin lsolation a n d chemical properties

Due to the continuing concern over environmental hazards, a college level biochemistry laboratory experiment has been developed which demonstrates the adverse effects of excess nitrate and nitrite in the environment. These pollutants can interfere with the human respiratory system hy interaction with the oxygen-carrying protein, hemoglohin. This experiment demonstrates the colors of oxyhemoglohin, deoxyhemoglobin, methemoglobin, and nitrosylhemoglohin and their visible absorption spectra. The rate of reaction of oxyhemoglobin with nitrite is followed spectro~hotometrically.Also, the experiment illustrates the effect of salt concentration on the integrity of red blood cells.

His

Background Hemoglohin 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 carhon dioxide hack to the lungs. The mechanism for Oz and COz transport are entirelv different. however. Because of its physihogical import&ce, hemoglobin has been exteusively studied. This has been facilitated hv the amwle sunwlv of starting material, the high concentration of hkmogl&n in red blood cells, and the ease with which it can he crystallized. Hemoglohin is a conjugated protein of molecular weight 64,450 made up of about 10,000 atoms of hydrogen, carbon, nitrogen, oxygen, and sulfur plus 4 iron(II) ions. The heme group consists of an iron(I1) a t the center of a porphyrin molecule. Each heme group is encircled by a chain of amino acids, one of 4 chains that together comprise the molecule of hemoglobin. The 4 chains of the protein portion of the molecule are called glohin (also called apohemoglobin). The 4 chains of glohin consist of two identical alpha chains and two identical beta chains. Heme alone will not hind oxygen; yet the specific combination of heme with glohin enables the 4 iron(lI) ions within the molecule to actively take up oxygen. The heme group in hemoglohin is composed of the quadridentate ligand protoporphyrin IX and Fez+. The Fez+ ion has a coordination number of 6 with the ligands arranged in an octahedral complex as shown in Figure 1.The fifth coordination position is occupied by the imidazole group of a histidine in the globin portion of the molecule. The sixth position (marked X in Fieure 1) is taken hv molecular bxygen in oxyhemoglohin a n d by NO & nitrosylhemoglohin. In methemoglobin (Fe3+) the sixth ligand site is-occupied by a wate~molecule.x:ray studies have shown that crystals of the liganded form are isomorphous. In contrast, the ligand-free structure, deoxyhemoglobiu, is crystallographically different from the liaanded hemoglohin forms mentioned previously and appears to be unique ( I ) . The absence of a ligand in the sixth coordination position has not been observed in deoxyhemoglohin itself. However, this has been inferred for deoxyhemoglobin from the observed absence of a ligaod in the related molecule deoxymyoglohin (2). There are 2 important health problems concerning hePresented in part at the 162nd American Chemical Society Meeting held in Washington D. C., September 1971.

X Figure 1 Hemachrome.

moglohin. The toxicity of carhon monoxide is due chiefly to the formation of carbonyl hemoglohin (also called carbon monoxide hemoglohin or carhoxyhemoglohin). Carbon monoxide adds on to the heme iron as does oxygen, but with greater affinity. The reactions involved are as follows 0,

CO HbO,

+

+

+

HbO, Hb --' HKO CO ==== HbCO

Hb

(1)

==+

+

(2)

O?

(3)

where Hh, HhOz, and HbCO represent deoxyhemoglohin, oxyhemoglohin, and carhonylhemoglohin, respectively. The equilibrium constant (3) for reaction (3) is

When hemoglobin is exwosed to a mixture of eases which contain ~ 0 ; t h e r e is competition for the hemoglohin with the equilibrium favoring formation of carhoxyhemoglobin. If a person poisoned hy breathing air containing CO is given pure 02, the conversion of carbon monoxide hemoglobin to oxyhemoglobin will he promoted as a result of La Chatelier's principle. Another health problem concerns the oxidation of hemoglobin to methemoglobin which is brown in color. This can he accomplished by the addition of nitrite to oxvhemoglobin, and the kinetics of the reaction are conveniently followed spectrophotometrically. Because of this reaction there is concern about nitrite in the environment. The pmblem has its inception with nitrate pollution in rural communities since heavy use of nitrate fertilizer can eventually lead to nitrate in the water supply. The reduction of nitrate to nitrite can then he accomplished by hacteria in the stomach. The result is a disease called methemoglohinemia which is particularly hazardous for infants and is sometimes fatal. There are several reasons why this disorder is more prevalent among infants than in adults. First, the infant's total fluid intake per body weight is approximately three times that of an adult (4). Second, the Volume 50,Number 5, May 1973 / 347

child's inability t o produce enough gastric acid results i n a higher pH i n t h e gastrointestinal t r a c t i n which nitratereducing bacteria can exist (5). Third, fetal hemoglobin is more easily oxidized t h a n is adult hemoglobin (6). Fourth, there is decreased activitv i n t h e infant of t h e enzyme responsible for normal methemoglobin reduction (71. Because of this problem t h e U. S. Public Health Service h a s set t h e limit b f nitrate i n drinking water a t 45 ppm. An article has appeared i n this J o u r n a l describing t h e analysis for nitrate by t h e hrucine method (8). Nitrite is also of environmental concern since i t is added to meats t o maintain the red color a n d t o a c t a s a preservative. T h e reader m a y h e puzzled a s t o why this is done since a s previously mentioned nitrite reacts with oxyhemoglohin t o produce methemoglobin which is brown i n color. However, if nitrite is added t o a solution containing oxyhemoglobin a n d a reducing agent such a s ascorbic acid, a red nitrosylhemoglohin (also called nitrosohemoelohin or nitric oxide hemoglobin) is produced. T h e meat industry tries t o maintain