Robert C. Terwilliger

The effect of organic acids on oxygen binding of hemocyanin from the crab Cancer magister

Abstract 1. 1. l -lactate raises the oxygen affinity of both stripped and unstripped hemocyanin (Hc) of Cancer magister . The effect of lactate is independent of pH in the range 6.8-8.3. 2. 2. d -lactate, glycolate and 2-methyl lactate also increase the Hc oxygen affinity, but to a lesser extent than l -lactate. Other structural analogues such as acetate, alanine, α-hydroxybutyrate, pyruvate and propionate have no effect on oxygen binding. 3. 3. These results suggest that C. magister Hc has an allosteric site with high specificity for L-lactate. 4. 4. The amount of lactate and H + in the blood of an exercised C. magister explains the change in Hc oxygen affinity brought about by high levels of locomotor activity. 5. 5. However, the amount of lactate in the blood of a resting crab does not explain the difference in Hc oxygen affinity between stripped and unstripped hemocyanin. 6. 6. End-products of anaerobic metabolism found in other taxa do not affect C. magister Hc oxygen binding. 7. 7. Thus, selection for an oxygen carrier with properties that respond to a particular form of anaerobic metabolism appears to have occurred. 8. 8. These findings confirm an earlier suggestion that an unidentified, low molecular weight factor is also present in crab hemolymph. 9. 9. The respiratory importance of the lactate effect following maximal locomotor activity is its enhancement of O 2 uptake at the gill rather than conservation of venous reserve at the tissues.

The quaternary structure of a molluscan (Helisoma trivolvis) extracellular hemoglobin.

The hemoglobin (erythrocruorin) of the planorbid mollusc Helisoma trivolvis has a molecular weight of 1.7-10(6) and a sedimentation coefficient (s0 20, w) of 33.8 S at pH 7.0. At pH 2.0, the pigment consists of 32 S and 13 S material. The hemoglobin exists as a 350 000 molecular weight submultiple in 6 M guanidine and can be further dissociated into a 175-200 000 dalton polypeptide in 6M guanidine, 0.1 M 2-mercaptoethanol or by sodium dodecyl sulfate gel electrophoresis of globin, performic acid oxidized globin or carboxymethylated globin. Electron microscope observations show a ten-membered ring structure measuring 200 A in diameter. It is proposed that Helisoma hemoglobin consists of a 1.7-10(6) dalton circular assembly of ten 175-200 000 dalton polypeptide chains. The amino acid composition of the pigment is reported. The hemoglobin contains one heme per 18-19 000 g protein. Limited proteolysis of the intact pigment shows 60 000, 40 000 and 17 000-18 500 dalton components when analyzed by sodium dodecylsulfate gel electrophoresis. It is likely that the 175-200 000 dalton polypeptide consists of a linear arrangement of 8-12 heme-containing domains, each domain having a molecular weight of 18-19 000.

Thermal Vent Clam (Calyptogena magnifica) Hemoglobin

A heterodont bivalve mollusk Calyptogena magnifica, from the East Pacific Rise and the Gal�pagos Rift hydrothermal vent areas, contains abundant hemoglobin in circulating erythrocytes. No other known heterodont clam contains a circulating intracellular hemoglobin. The hemoglobin is tetrameric and has a relatively high oxygen affinity, which varies only slightly between 2� and 10�C. The presence of hemoglobin in the clam may facilitate the transport of oxygen to be used in chemoautotrophic hydrogen sulfide metabolism.

The structure of hemoglobin from an unusual deep sea worm (Vestimentifera)

Abstract 1. 1. The extracellular hemoglobin from a species of Vestimentifera, obtained from hydrothermal vents at 2.5 km depths of the ocean, has been studied. 2. 2. The hemoglobin molecules appear as two-tiered hexagonal structures in the electron microscope. The hexagons measure 160 A high and 245 A wide. 3. 3. The pigment, which was purified from frozen specimens, appears to be very unstable and elutes on Sepharose 4B as aggregates of approx 1.7 × 10 6 and 400,000 mol. wt. 4. 4. The subunit molecular weight of purified hemoglobin, determined by sodium dodecyl sulfate gel electrophoresis, shows heterogeneity with molecular weights around 15,000 and 30,000. 5. 5. The purified hemoglobin contains one heme per 23,000 g protein. The amino acid compositions of isolated fractions are presented. 6. 6. This Vestimentifera, which shows characteristics of both the phylum Pogonophora as well as Annelida, has an extracellular hemoglobin whose structure is very similar to that of annelid extracellular hemoglobins.

Comparison of chlorocruorin and annelid hemoglobin quaternary structures

Abstract 1. 1. The structures of Eudistylia vancouveri chlorocruorin and Pista pacifica and Thelepus crispus extracellular hemoglobins have been examined by electron microscopy. The intact pigments display a two-tiered hexagonal array characteristic of other annelid high molecular weight hemoglobins and chlorocruorins. 2. 2. The smallest subunit molecular weight of these pigments determined by SDS gel electrophoresis is 14–15,000; Pista hemoglobin contains some additional 30,000 molecular weight material under a variety of denaturing conditions. Earthworm hemoglobin is also heterogenous upon SDS gel electrophoresis, consisting of at least six components with mol. wt ranging from 13, 000 to 35,000. 3. 3. The amino acid compositions of these high molecular weight pigments are compared with one another and with the intracellular hemoglobins of other invertebrates and vertebrates. It is likely that Eudistylia chlorocruorin and certain high molecular weight annelid hemoglobins form a group of closely related proteins.

Erythrocruorins of Euzonus mucronata treadwell: Evidence for a dimeric annelid extracellular hemoglobin

Abstract 1. 1. The extracellular hemoglobin of the opheliid polychaete Euzonus mucronata is unusual in that it consists of a 3.2 × 10 6 as well as a 6–7 × 10 6 molecular weight hemoglobin at pH 7.O. 2. 2. Electron microscopy of the 3.2 x 10 6 dalton material shows the typical annelid hemoglobin pattern of a two-tiered hexagonal array of submultiples whereas the 6–7 × 10 6 dalton pigment has a four-tiered hexagonal arrangement, a putative dimer of a 3.2 × 10 6 molecular weight material. A few six-tiered trimeric stacks are also present. 3. 3. The dimeric material, which represents 10–15% of the total pigment, is present in unpurified vascular fluid as well as in purified hemoglobin. The constant stoichiometry between the 3.2 × 10 6 dalton monomer and the dimeric pigment under different methods of purification as well as in hemoglobin examined directly from the worm suggests that the dimer is not a preparative artifact. The dimer is not in a state of rapid dissociation-association equilibrium with the monomer nor does the dimer seem to be the result of a ligand-linked association of the 3.2 × 10 6 dalton monomer. 4. 4. The subunit molecular weights of both the monomer and dimer are the same with polypeptide chains of 36,700 ± 800, 31,400 ± 800. 14,400 ± 300. and 12,500 ± 300 as determined by sodium dodecyl sulfate gel electrophoresis. The same molecular weight heterogeneity is present for globin which has been performic acid oxidized, carboxymethylated in 7–8 M guanidine HC1 and 0.1 M in 2-mercaptoethanol, or treated with 8 M urea, 0.1 M in 2-mercaptoethanol. 5. 5. The hemoglobin contains one heme per 22,400 g protein. The amino acid composition of this protein is presented.

Quarternary structure of Pista pacifica vascular hemoglobin

Abstract 1. 1. The chemical and physical properties of the vascular hemoglobin of the terebellid polychaete Pista pacifica have been studied. 2. 2. The pigment has a sedimentation coefficint ( s 20, w 0 ) of 58 and a molecular weight of 3·4 × 10 6 at neutral pH in 0.01 M MgCl2. 3. 3. The hemoglobin dissociates at neutral pH in the presence of 0·01 M EDTA into submultiples of molecular weight ca. 280,000; further dissociation into 120,000 and 60,000 molecular weight components is observed at alkaline pH. The smallest subunit determined by SDS gel electrophoresis has a molecular weight of 15,500±500 . 4. 4. Co-operative oxygen binding similar to that found in the intact polymer exists at the 280,000 molecular weight submultiple level. 5. 5. The structure and amino acid composition of the vascular hemoglobin of Pista pacifica are compared with hemoglobins of other annelids and with the coelomic cell hemoglobins of Pista.

Subunit heterogeneity of cancer magister hemocyanin

Hemocyanin from the Dungeness crab, Cancer magister, has been described as a 25-S two-hexamer assembly of two different 5-S subunits. We have found that at least six different 5-S polypeptide chains constitute this hemocyanin. They can be separated from one another by sodium dodecyl sulfate slab gel electrophoresis as well as by regular gel electrophoresis. The six 5-S polypeptides appear very different from one another when each SDS-treated subunit is partially digested with Staphylococcus aureus V8 protease. This pattern of six subunits is present both in hemolymph which has been examined immediately upon removal from the animal as well as in hemocyanin which has remained at room temperature for two weeks. Thus, it is unlikely that the heterogeneity is a result of proteolysis during preparation of the sample. Possible implications of the high degree of subunit heterogeneity on the proteins quaternary structure are discussed.

Molecular weight of Eudistylia vancouveri chlorocruorin and its subunits.

The chlorocruorin of the marine polychaete Eudistylia vancouveri has a molecular weight of 3.1-10(6) and a sedimentation coefficient (S020, w) of about 57 S at pH 8.0 in the presence of 0.01 M Mg2+. The quaternary structure of this pigment is unaffected by pH between 6.0 and 11.5 in the presence of 0.01 M Mg2+ whereas in 0l01 M EDTA, the pigment begins to dissociate above pH 9.0 into smaller submultiples. The chlorocruorin can be converted into subunits with molecular weights of about 14 000-15 000 and 30 000 as determined by sodium dodecyl sulfate-gel electrophoresis and 14 000-15 000 as measured by gel chromatography of the carboxy-methylated derivative in 8 M urea, 0.1 M 2-mercaptoethanol, or by sedimentation equilibrium in 6 M guanidine-HCl and 0.1 M 2-mercaptoethanol. The pigment contains 0.212 +/- 0.008% iron corresponding to 1 g atom iron per 26 300 g chlorocruorin. The amino acid composition of this pigment is reported. The subunit structure of Eudistylia chlorocruorin and the polymeric annelid hemoglobins are similar in many respects.

Structural studies of a branchiopod crustacean (Lepidurus bilobatus) extracellular hemoglobin. Evidence for oxygen-binding domains.

The extracellular hemoglobin of the notostracan branchiopod Lepidurus bilobatus has an apparent molecular weight of 680,000 and may exist in a dissociation-association equilibrium dependent on pH and ligand state. The pigment contains one heme per 18,000 g protein. However, attempts to dissociate the hemoglobin by harsh denaturing conditions results in a 33-34,000 molecular weight polypeptide chain as well as traces of some 62-64,000 molecular weight material. Limited proteolysis of this hemoglobin with subtilisin produces 14,800 and 16,500 dalton heme-containing polypeptides (domains) which bind oxygen reversibly. These domains, isolated by column chromatography, have a heme content similar to the intact pigment. It is proposed that the intact 34,000 dalton subunit of Lepidurus hemoglobin consists of two linearly linked oxygen binding domains. Oxygen binding properties of the intact hemoglobin show a low oxygen affinity with a slight Bohr effect. In contrast, the isolated domains display a relatively high oxygen affinity and lack a Bohr effect between pH 7.0 and 8.0. It is apparent that the intact 34,000 dalton polypeptide is necessary for the expression of the heterotropic interactions of the native pigment.