Physical properties and subunits of Dina dubia erythrocruorin

Abstract

The erythrocruorin of the freshwater leech Dina dubia possessed an s 20,w of 61 S and exhibited a slightly sigmoid oxygenation curve with n ⋍ 1.6 and P 50 = 2.4 mm at pH 7.4. A minimum mol. wt of 23 000 ± 2100 per heme group was determined from the iron and heme contents, 0.22 ± 0.02 and 2.92 ± 0.35 weight %. The subunit composition of this erythrocruorin was investigated using polyacrylamide gel electrophoresis and gel filtration in sodium dodecyl sulfate at neutral pH and gel filtration at pH 9. Sodium dodecyl sulfate electrophoresis of Dina erythrocruorin revealed the presence of five subunits (1–5) with mol. wts of about 13 000, 21 000, 23 000, 25 000 and 31 000, respectively. When the erythrocruorin was reduced with mercaptoethanol prior to dodecyl sulfate electrophoresis, three subunits (I–III) were observed, two possessing molecular weights in the range 12 000–14 000 (I and II) and one possessing a molecular weight of about 28 000. One of the subunits I, II, was provided by the dissociation of the 31 000 subunit. Subunit III (28 000) consisted of subunits 2, 3, and 4. It is likely that not all of the polypeptide chains constituting Dina erythrocruorin are associated each with a heme group.