SOME PHYSICAL CHEMICAL EXPERIMENTS WITH HEMOGLOBINS FROM NORMAL AND GENETICALLY ANEMIC MICE

Abstract

Hemoglobins of normal mice and of mice carrying the W series of alleles conditioning an anemia and abnormalities in pigmentation and germ cell development were tested for possible physical chemical differences. The animals used were genetically identical except for the W-locus, and 15 sets of adult Wv Wv 1 Wvw and ww littermates, ranging from 2 1/2 to 9 months in age and of the same sex where possible, were accumulated to provide enough blood for the tests. Blood from the same genotypes was pooled and diluted with an equal volume of Alsever''s soln. to prevent clotting. The hemoglobins were prepared by centrifuging the blood samples, removing the supernatants from the pellets and suspending the pellets in cold 0.9% NaCl. After being centrifuged and washed three times 0.2 ml. distilled water and 1 ml. toluene were added to each pellet, carbon monoxide was bubbled through the suspension and the cells allowed to lyse overnight at 4[degree]C. The suspensions were centrifuged and the clear red aqueous layers drawn off by syringe, then stored at 4[degree]C. In order to provide a sensitive test for electrophoretic differences among hemoglobins, solns. of carbonmonoxyhemoglobin (5 mg/ml) were prepared by diluting the stock solns. with cacodylate-NaCl buffer, and dialyzed overnight against the same buffer saturated with carbon monoxide. Electrophoresis was performed in a Klett Tiselius apparatus at 2[degree] C at a field strength of 8.1 volts/cm. for 12,000 seconds. Electrophoretic patterns and mobilities were almost identical for hemoglobins from ww, Wv w and Wv Wv genotypes and from an equal part mixture of ww and Wv Wv. Sedimentation and solubility experiments for ww and Wv Wv mice showed that these genotypes were similar. These results indicate that the hemoglobins of ww, Wv w and Wv Wv mice are very similar or identical.