Hemoglobin unstable

Many of the substitutions mentioned above adversely affect the stability of the tetrameric molecule and cause easy denaturation and precipitation within erythrocytes to form Heinz bodies. Deletion of one or more amino acids is also likely to cause instability, preeipitation (e.g., Hbs Leiden, Tochigi, Tours, Gun Hill), and membrane damage. Consequently, these variants are associated with intravascular hemolysis, anemia, reticulocytosis, splenomegaly, and, in some patients, intermittent urobilinuria. 

The difference in disease severity among the unstable hemoglobins may reside in the number and time of 

Hemichromes precipitate and form Heinz bodies, which attach hydrophobically to the erythrocyte membrane, increasing membrane permeability and the rate of lysis. Loss of heme and oxidation of sulfhydryl groups do not appear to be necessary for precipitation. 

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A search for Heinz bodies Is helpful In the detection of an unstable hemoglobin, of a-thalassemia, homozygous 3-thalas-semla and related abnormalities, because hemoglobin often precipitates In the red cells of patients with one of these disorders. Inclusion bodies may consist of precipitated unstable hemoglobin, of 3 chalns (In a-thalassemia), or of a chains (In thalassemia) ... 

Other tests useful for the detection of unstable hemoglobins are the heat stability test, the Isopropanol precipitation test, and the PCMB or PMB precipitation test ... 

Unstable hemoglobin variants are often found In persons with unexplained hemolytic anemias. Below are some guidelines for their detection. 

Often unstable hemoglobins have a decreased number of heme groups. This number can be calculated from the optical densities of a solution of the cyanferrl derivative of the Isolated variant at 540 and at 280 nm using normal Hb-A as control. 

Appreciable amounts of Hb-F can be present in patients with sickle cell anemia, with SC disease, with CC disease, with the Hb-Lepore disorders, and with hemolytic anemia due to the presence of an unstable hemoglobin variant [(A9, B21, B37, G6, H15, Jl, L2, N9, R15, Sll, S33, S49, T3) and others]. 

Fig. 19. The heterogeneity of fetal hemoglobin in subjects with various hemoglobinopathies. Group A represents SS patients of which the parents have been diagnosed as Hb-S heterozygotes such information is incomplete for subjects of groups B + C. The group labeled other concerns patients with Hb-Richmond, Sabine, other unstable hemoglobins, and Hb-Lepore. Boxed-in values are from relatives.

The presence of an unstable hemoglobin variant often leads to the occurrence of Heinz bodies in peripheral blood after incubation with a redox dye, particularly in a carrier who has been splenectomized. Three procedures are recommended ... 

Fig. 20. The formation of Heinz bodies in a subject with the unstable hemoglobin Louisville. The patient was splenectomized at the age of 21 years. From Keeling et al. (K9) with permission of the authors and publisher. 

It has been shown that the stability of the heme group is one of the most important factors in the denaturation (and precipitation) of hemoglobin both in vivo and in vitro (R17). Thus, when the replacement of an amino acid residue in the chain occurs in a position which is part of the heme contact, the stability of the molecule is usually affected, resulting in loss of the heme group from that particular chain and in a progressive denaturation. Rapid denaturation, therefore, is a characteristic of an unstable hemoglobin (R20). Precipitation of the protein takes place within the cell and intraerythrocytic inclusion bodies appear leading to increased cell destruction. 

This rapid denaturation of hemoglobin affords this method for determining the presence of an unstable hemoglobin variant even when starch gel electrophoresis of hemolysate shows no evidence of an abnormality. The heat stability of the hemoglobin variant is compared with that of a normal control the two are incubated simultaneously in a phosphate buffer at 60°C (K9). 

J5. Jacob, H. S., and Winterhalter, K. H., The role of hemoglobin heme loss in Heinz body formation studies with a partially heme-deficient hemoglobin and with genetically unstable hemoglobins. J. Clin. Ineest. 49, 2008-2016 (1970). 

Rll. Ranney, H. M., Jacobs, A. S., Udem, L., and Zalusky, R., Hemoglobin Riverdale-Bronx an unstable hemoglobin resulting from the substitution of arginine for glycine at helical residue B6 of the /3 polypeptide chain. Biochem. Biophys. Res. Commun. 33, 1004-1011 (1968). 

Zinkham WH. Unstable hemoglobins and the selective hemolytic action of sulfonamides. Arch Intern Med 1977 137(10) 1365-6. 

The y- and 5-chain mutants are difficult to study because of the small fraction of HbF and HbA2 present in adult erythrocytes. Overt clinical symptoms associated with y- and 5-chain variants are rare. By routine screening, 14 5-globin variants have been discovered but are of no clinical consequence. Thirty-five mutant y sequences (involving the Gy or the Ay chains) have been identified. They are all benign except for HbF Poole [Gyl30(H8) Tyr —> Gly], an unstable hemoglobin that causes hemolytic disease in the newborn. 

HbH and many unstable hemoglobins spontaneously precipitate within the red cells, forming Heinz bodies, which can be detected in splenectomized patients by staining with methylene blue. Alternatively, precipitation of these hemoglobins can be induced and the precipitates visualized by incubation of the erythrocytes with a redox dye such as brilliant cresyl blue (Chapter 28). 

Many abnormal hemoglobins are also much more readily denatured by heat than normal hemoglobins. Heating an unstable hemoglobin for 30 minutes at 60°C usually causes complete denaturation, whereas HbA is... 

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