Hemocyanin of the chiton, Stenoplax conspicua (Dall)

1. The hemocyanin of the chiton, Stenoplax conspicua, has a molecular weight determined by light-scattering of 4.2 X 10(6) daltons, (dt) and a sedimentation coefficient of 60 S. 2. The fully dissociated subunits in 6.0 and 8.0 M urea, and at pH 8.9-10 in the absence of divalent ions, have molecular weights of 4.15-4.30 x 10(5) and 4.17-4.75 x 10(5) dt, which is close to one-tenth of the molecular weight of the parent hemocyanin assembly. 3. The pH dependence of the molecular weights from pH 4.5 to 11 exhibit bell-shaped transition profiles, best accounted for by a three-species, decamer to dimer to monomer scheme of subunit dissociation, with one acidic and one basic ionizing group per dimer and 5-8 acidic and basic groups per monomer. 4. In the absence of stabilizing divalent ions S. conspicua hemocyanin is relatively unstable. At pH 7.4 in the presence of 0.01 M EDTA, it is predominantly in the dimeric state, characterized by a sedimentation constant of 18 S. It is also more readily dissociated to monomers at high pHs (8-9 and above) than are the C. stelleri and A. granulata hemocyanins. 5. Urea and GdmCl are effective dissociating agents of S. conspicua hemocyanin. The urea dissociation profile obtained at pH 8.5, 0.01 M Mg2+, 0.01 M Ca2+, and analyzed by means of the decamer-dimer-monomer scheme of subunit dissociation gave estimates of about 30 amino acid groups (Napp) at the dimer contacts within the hemocyanin decamers and about 120 groups per monomer within each dimer, suggesting hydrophobic stabilization of hemocyanin assembly.     

Physical studies of the hemocyanin of the marine gastropod, Kelletia kelleti (Forbes).

1. The hemocyanin of the Californian whelk, Kelletia kelleti, investigated at pH and ionic conditions close to physiological, has a molecular weight close to 9.0 x 10(6) and a sedimentation constant of 114S, characteristic of the di-decameric structure of molluscan hemocyanins. Light-scattering measurements at pH 8.0, 0.05 M Mg2+, 0.01 M Ca2+ gave a molecular weight of 9.0 +/- 0.6 x 10(6), and scanning transmission electron microscopy produced nearly the same particle mass of 9.22 +/- 0.50 x 10(6) daltons (Da). 2. Light-scattering measurements on the fully dissociated monomers in the presence of 8.0 M urea and at pHs 10.6 and 11.0 gave molecular weights of 4.50 x 10(5)-4.91 x 10(5), that are close to one-twentieth of the mass of the parent di-decameric hemocyanin assembly. 3. Changes in pH produced a bell-shaped molecular weight profile, with molecular weights close to 9.0 x 10(6) in the pH region of about 5.5-8.0, and progressive dissociation to 4.5 x 10(5) Da monomers in the region below pH 4.0 and above pH 9.0 or 10, depending on the absence or presence of stabilizing Mg2+ ions (0.01 M). 4. In the absence of divalent ions some aggregation of hemocyanin was found at pHs close to 5.0, with observed molecular weights above 10 x 10(6) (investigated at a hemocyanin concentration of 0.10 g/l). The early studies of Condie and Langer (Science 144, 1138-1140, 1964) had shown that Kelletia kelleti hemocynanin aggregates at acidic pHs close to the isoelectric point, forming linear polymers of the hemocyanin di-decamers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical investigations of the hemocyanin of the chiton, Cryptochiton stelleri (Middendorff)

The hemocyanin of the giant Pacific chiton, Cryptochiton stelleri has a molecular weight of 4.2 +/- 0.3 X 10(6), determined by light-scattering, and a sedimentation coefficient of 60S. The fully dissociated subunits in nondenaturing solvents, at pH 10.6, 1 X 10(-2)M EDTA and in 8.0 M urea, pH 7.4 have molecular weights of 4.10 X 10(5) and 4.35 X 10(5), close to one-tenth of the molecular mass of the parent hemocyanin decamers. In the pH region from about 3.5 to 11 the molecular weight (Mw), determined at constant protein concentration of 0.10 g1(-1) exhibits a bell-shaped molecular weight profile centering about the physiological pH of the hemolymph of 7.2. The pH-Mw profile is best accounted for in terms of a three state, decamer-dimer-monomer dissociation scheme. Analysis of the Mg2+ and Ca2+ effects on the molecular weight transitions suggest stabilization of the hemocyanin decamers through one bound divalent ion per hemocyanin monomer or dimer. Urea, GdmCl, and the higher members of the chaotropic salt series are effective dissociating agents for Cryptochiton stelleri hemocyanin. The dissociation profile obtained with urea at pH 8.5, 0.01 M Mg2+, 0.01 M Ca2+ has been analyzed in terms of both the two- and three-species schemes of subunit-dissociation. Hydrophobic stabilization of the subunit contacts is suggested by the large number of apparent amino acid groups (Napp), of the order of 30 between dimers stabilizing the decamers, and 120 apparent amino acid groups between each monomer forming the constituent dimers.     

Light-scattering and scanning transmission electron microscopic investigation of the hemocyanin of the bivalve, Yoldia limatula (Say)

1. The hemocyanin of the bivalve, Yoldia limatula (Say) was found by light-scattering to have a mol. wt of 8.0 +/- 0.6 x 10(6). Mass measurements by scanning transmission electron microscopy (STEM) gave a particle mass of 8.25 +/- 0.42 x 10(6) for the native particle and 4.09 +/- 0.20 x 10(6) for the half-molecule. 2. The hemocyanin subunits fully dissociated in 8.0 M urea and 6.0 M GdmCl at pH 8.0, and at pH 11.0, 0.01 M EDTA have mol. wts of 4.38 x 10(5), 4.22 x 10(5) and 4.71 x 10(5), close to one-twentieth of the parent molecular weight of Y. limatula hemocyanin and most gastropod hemocyanins. 3. Analyses of the urea dissociation transitions studied at pH 8.0, 1 x 10(-2) M Mg2+, 1 x 10(-2) M Ca2+ and pH 8.0, 3 x 10(-3) M Ca2+ suggest few hydrophobic amino acid groups, of the order of 10 to 15 at the contact areas of each half-molecule or decamer. 4. The further dissociation of the decamers to dimers and the dimers to monomers indicates the presence of a larger number of amino acid groups of ca 35-40/dimer and 100-120/monomer. 5. This suggests hydrophobic stabilization of the dimer to dimer and monomer to monomer contacts within the decamers, as observed with other molluscan hemocyanins.     

The hemocyanin of the ramshorn snail, Marisa cornuarietis (Linné)

1. The hemocyanin of the freshwater snail, Marisa cornuarietis exists predominantly as a di-decamer with the approximate mol. wt of 8.5 x 10(6) and a sedimentation coefficient of 100 S. Sedimentation and scanning transmission electron microscopy experiments indicate that about 15-20% of the hemocyanin forms tri-decameric and possibly higher aggregates with mol. wts of 12.5 x 10(6) and 130 S. 2. The fully dissociated subunits in 8.0 M urea and 6.0 M GdmCl have mol. wts of 4.1 to 4.7 x 10(5) which is close to one-twentieth of the major di-decameric component of the native hemocyanin. 3. Subunit dissociation by the urea series and the Hofmeister salt series of reagents suggests hydrophobic stabilization of the decamers or half-molecules of the parent hemocyanin. As with the other molluscan hemocyanins the order of effectiveness of the ureas as dissociating agents shows increased efficacy with increasing hydrophobicity or chain-length of the urea substituents. 4. Denaturation of the hemocyanin subunits by the ureas and Hofmeister salt series, investigated by circular dichroism measurements, essentially follow the same trend in effectiveness as observed by changes in subunit dissociation followed by light-scattering mol. wt measurements. 5. The observed denaturation transitions are shifted to much higher ranges of reagent concentration than the concentrations required for the dissociation of the hemocyanin subunits.     

The hemoglobin of the aquatic snail, Planorbella duryi (Wetherby)

1. The hemoglobin of the pond snail, Planorbella duryi has a molecular weight of 1.64 x 10(6) to 1.77 x 10(6) as determined by light-scattering at 630 nm and a sedimentation coefficient of 36 S. 2. The analysis of the circular dichroism spectrum obtained in the 190-250 nm region suggests a high degree of helical folding of the polypeptide chains of P. duryi hemoglobin analogous to human hemoglobin and myoglobin, with estimates of alpha-helical folding of about 60-65%, 0-5% beta-structure, and the remaining portion of the chains in unordered form. 3. The dissociated subunits in 6.0 M GdmCl, in the absence and in the presence of reducing reagent (0.1 M dithiothreitol), have a molecular weight of 3.73 +/- 0.23 x 10(5) and 1.93 +/- 0.04 x 10(5), suggesting a di-decameric assembly of the parent hemoglobin organized in the form of five dimers held together by disulfide-linkages. 4. The native hemoglobin is strongly resistant to both pH dissociation and dissociation by urea and such salts as NaCl and NaClO4. Dissociation and denaturation could only be effected in concentrated GdmCl solutions. 5. The influence of the various dissociating agents on the quaternary structure suggest ionic stabilization of the decameric assembly, which is stabilized by salt bridges between the subunits.     

Hemocyanin of the chiton Acanthopleura granulata

The subunit structure and solution conformation of the hemocyanin of the chiton Acanthopleura granulata were investigated by light-scattering, ultracentrifugation, viscosity, absorbance, and circular dichroism methods. The molecular weight, determined by light scattering at pH 7.4 in the presence of 0.05 M Mg2+ and 0.01 M Ca2+, was (4.2 +/- 0.3) X 10(6), while those of dissociated subunits in the presence of 8.0 M urea (at pH 7.4) and at pH 10.7 were found to be 4.57 X 10(5) and 4.58 X 10(5), respectively. Circular dichroism and absorbance measurements at 222 and 346 nm indicate only minor changes in the conformation of the folded domains of the hemocyanin subunits in these dissociating solvents. As with the hemocyanins of the snails Busycon canaliculatum, Lunatia heros, and Littorina littorea, exposure to 4.0-6.0 M guanidinium chloride (GdmCl) is found to produce unfolding of the domains, resulting in much more pronounced spectral changes and a further drop in molecular weight. A Mw of 3.2 X 10(5) was obtained with Acanthopleura hemocyanin in 6.0 M GdmCl, suggesting hidden breaks in the polypeptide chains analogous to those observed with the gastropodan hemocyanins. Both urea and pH dissociation showed gradual declines in the molecular weights, consistent with a decamer-dimer-monomer scheme of subunit dissociation. The bell-shaped molecular weight profiles obtained in the pH region from 5 to 11 can be accounted for by assuming two proton-linked groups per dimer, characterized by apparent pK values of 5.5 and 9.5, and the further involvement of five to eight acidic and five to eight basic groups per monomer, having apparent pK values of 5.0 and 10.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilizing influence of calcium and magnesium ions on the decameric structure of chiton hemocyanins

The stabilizing effects of Ca2+ and Mg2+ ions on the decameric structure of hemocyanins from two representative chitons, Stenoplax conspicua and Mopalia muscosa were investigated by light-scattering molecular weight measurements, ultracentrifugation, absorbance, and circular dichroism methods. The dissociation profiles at any given pH resulting from the decrease in divalent ion concentration, investigated at a fixed protein concentration of 0.1 g.liter-1, could be fitted by a decamer-to-dimer-to monomer scheme of subunit dissociation. The initial decline in the light-scattering molecular weight curves required one or two apparent binding sites per hemocyanin dimer formed as intermediate dissociation product, with apparent dissociation constants (kD,2) for Ca2+ ions of 0.7 to 7 X 10(-4) M, not very different from the value of 2.5 X 10(-4) M obtained by Makino by equilibrium dialysis for the hemocyanin of the opistobranch, Dolabella auricularia. The binding of Mg2+ ion to S. conspicua and M. muscosa hemocyanins appears to be both weaker than the binding of Ca2+ and more pH dependent, with kD,2 values ranging from the 3 X 10(-4) to 4 X 10(-2) M at pH 8.5 to 9.5. The dissociation the decameric hemocyanin species (sedimentation coefficient ca. 60 S) is also observed in the ultracentrifugation with the initial appearance of 18-20 S dimers, followed by a shift in equilibrium to monomeric species of lower sedimentation rates of 11-12 S as the divalent ion concentration is reduced below 1 X 10(-4) M Ca2+ and Mg2+. The dissociation of dimers to monomers in the second step of the reaction is characterized by one or two binding sites per subunit and a somewhat stronger affinity for divalent ions, indicated by apparent dissociation constants (kD,1) of 0.7 X 10(-4) to 3 X 10(-3) M. Circular dichroism and absorbance measurements at 222 and 346 nm suggest no significant changes in the conformation of the hemocyanin subunits produced by the different stages of subunit dissociation.     

Subunit dissociation of Busycon canaliculatum hemocyanin

The hemocyanin of the channeled whelk, Busycon canaliculatum, is a multisubunit protein with a molecular weight close to 9 X 10(6). The increase in pH above neutrality and the addition of 0-5 M urea and 0-2 M GdnHCl is found to dissociate the whole molecules to half-molecules and smaller dimeric and monomeric fragments of one-tenth and one-twentieth mass of the parent hemocyanin. The molecular weight transitions investigated at constant protein concentration of 5 X 10(-2) g X l-1 show no clearly discernible plateau regions, where essentially only half-molecules and one-tenth molecules are present. The ultracentrifugation patterns in much of the dissociation region produced by urea at pH 6.9 suggests the presence of three distinct components consisting of whole molecules, half-molecules and largely one-tenth molecular weight fragments. At pH 8.2 and higher, where whole molecules are largely absent, the effects of urea on the dissociation of half-molecules to tenths and tenth-molecules to twentieth molecule was investigated by means of light scattering. Analysis of the urea data based on a decamer to dimer and dimer to monomer scheme of dissociation used in our earlier studies gave apparent estimates of about 90 amino acid groups at the contact areas of the dimers in the half-molecules and 110 groups at the monomer contacts forming the dimers. The latter relatively large estimate of groups suggests that the dissociation of the tenth molecules or dimers must occur by longitudinal splitting of the contact areas along both the folded domains and the connecting chain segments of the twentieth molecules. Circular dichroism, absorbance and viscosity data suggest that the secondary structure and conformation of the folded domains of the hemocyanin subunits are largely retained at both high pH and in 3-8 M urea solutions. The molecular weights at pH 9.0-10.6 and in 3-8 M urea are found to be (4.2-4.7) X 10(5), close to one-twentieth of the mass of the parent hemocyanin. Denaturation and unfolding of the subunit domains is observed between 3 and 6 M GdnHCl solutions, as evidenced by the abolition of the characteristic copper absorbance in the neighborhood of 346 nm and the relatively pronounced changes in circular dichroism at 222 nm and intrinsic viscosity. The further decrease in molecular weights to about (2.6-3.2) X 10(5), below one-twentieth of the mass of hemocyanin suggests the presence of hidden breaks or scissions in the polypeptide chains suffered during isolation, which become exposed as a result of complete unfolding in GdnHCl solutions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Light-scattering investigation of the subunit structure and dissociation of octopoda hemocyanins

The molecular weights, subunit dissociation, and conformation in solution of the hemocyanins of three species of octopi were investigated by light-scattering, ultracentrifugation, absorbance, and circular dichroism methods. The molecular weights of the hemocyanins of Octopus bimaculoides, Octopus bimaculatus, and Octopus rubescens obtained by light scattering were 3.3 X 10(6), 3.4 X 10(6), and 3.5 (+/- 0.3) X 10(6), respectively. The average molecular weights of the fully dissociated hemocyanins of the same octopi, investigated at alkaline pH and in the presence of 8 M urea and 6 M guanidinium chloride (GdmCl), were found to be close to one-tenth of those of the parent proteins, with average molecular masses of 3.4 X 10(5), 3.3 X 10(5), and 3.3 (+/- 0.3) X 10(5). These findings confirm the earlier observations of van Holde and co-workers with other cephalopod hemocyanins that the basic cylindrical assembly of molluscan hemocyanins consists of 10 subunits. Circular dichroism and absorbance measurements suggest that the dissociated subunits at alkaline pH and in concentrated urea solutions retain their native, multidomain folding. Fairly concentrated GdmCl above 3-4 M is necessary to unfold fully the dissociated hemocyanin chains. Molecular weight measurements studied as a function of reagent concentration with the urea and Hofmeister salt series as dissociating agents show that the ureas are very effective dissociating agents, while the salts are ineffective to moderately effective reagents for octopus hemocyanin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recent aspects of the subunit organization and dissociation of hemocyanins

1. The hemocyanins of the arthropod phylum are built of multiples of hexamers consisting of 1,2,4,6 and 8 of such basic assemblies. Their molecular weights range from about 0.45 x 10(6) to 3.9 x 10(6) daltons. The basic hexameric unit consists of bean-shaped monomers organized in the form of two layers of trimers placed on top of one another. The subunits are heterogeneous, in most cases consisting of four or more electrophoretically different polypeptide chains. 2. Molluscan hemocyanins have an entirely different structure and pattern of assembly from the arthropodan hemocyanins. The basic assembly of the molluscan hemocyanins are decamers organized in the form of right-handed cylinders approximately 300 A in diameter and 140-190 A in height. Different species have one, two and sometimes more than two such assemblies forming correspondingly longer cylindrical particles with molecular weights ranging from about 3.3 x 10(6) to 13 x 10(6) daltons. Cephalopod and chiton hemocyanins consist of single decameric particles, while gastropods have hemocyanins organized of di-decamers or higher assemblies. The subunits of these hemocyanins are elongated protein chains with seven or eight folded globular domains, each housing a binuclear copper center capable of binding and delivering oxygen. 3. The dissociation behavior of the arthropod hemocyanin hexamers and di-hexamers with the hydrophobic urea series of reagents suggest polar and ionic interactions as the main sources of stabilization of the hexamers and the hexamer to hexamer contacts within the di-hexamers. 4. Dissociation studies with the same urea probes with the molluscan hemocyanins, however, suggest a different pattern of stabilization. The stabilization of the decamer to decamer contacts within the gastropod di-decamers appear to be predominantly polar and ionic with relatively few hydrophobic interaction sites. The dimer contacts within the decamers and the monomer to monomer contacts within the dimers observed in the octopus and chiton hemocyanins appear to be predominantly hydrophobic in nature. 5. The urea and the pH dissociation profiles of the single decameric assemblies of some of the octopus and chiton hemocyanins investigated by light-scattering molecular weight methods, have been fitted using either a two-species, decamer to dimer and decamer to monomer scheme of subunit dissociation or a three-species, decamer to dimer to monomer scheme of dissociation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Light-scattering investigation of the dissociation behavior of Lunatia heros and Littorina littorea hemocyanins

The subunit structure and dissociation of the hemocyanins of two marine snails, Lunatia heros and Littorina littorea, were investigated by light-scattering molecular weight methods. The hemocyanins of both species of snails are readily dissociated to fragments of one-tenth and one-twentieth of the parent proteins of close to 9 X 10(6) daltons by either increasing the pH or using dissociating reagents of the hydrophobic urea series or some of the Hofmeister salts. The lower members of the latter group of reagents, NaCl, and to some extent also NaBr were found to have only marginal effects on the observed molecular weight transitions, suggesting that the two hemocyanins investigated possess beta-type subunits, which are known to be resistant to NaCl dissociation. The molecular weight profiles obtained with the various dissociating reagents were single inverted sigmoidal-shaped curves for both Lunatia and Littorina hemocyanins, suggesting overlapping transitions. The ultracentrifugation patterns and the species-distribution plots based on the urea dissociation data of Littorina hemocyanin suggest the presence of whole, half, and one-tenth molecular weight species in the dissociation transition region. Fitting of the urea dissociation data of Littorina hemocyanin obtained at both pH 5.7 and pH 8.0, assuming a sequential two-step dissociation scheme used in our previous studies [Herskovits, T. T., & Russell, M. W. (1984) Biochemistry 23, 2812-2819], was found to be consistent with a model of a few hydrophobic binding sites at the contact areas of the half-molecules and a much larger apparent number of binding sites (Napp) at the side to side contacts of the one-tenth molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of D-arabinose isomerase purified from two strains of Escherichia coli

d-Arabinose isomerase (EC 5.3.1.3) has been isolated from l-fucose-induced cultures of Escherichia coli K-12 and d-arabinose-induced cultures of E. coli B/r. Both enzymes were homogeneous in an ultracentrifuge and migrated as single bands upon disc electrophoresis in acrylamide gels. The s(20,w) was 14.5 x 10(-13) sec for the E. coli K-12 enzyme and 14.3 x 10(-13) sec for the E. coli B/r enzyme. The molecular weight, determined by high-speed sedimentation equilibrium, was 3.55 +/- 0.06 x 10(5) for the E. coli K-12 enzyme and 3.42 +/- 0.04 x 10(5) for the enzyme isolated from E. coli B/r. Both enzyme preparations were active wth l-fucose or d-arabinose as substrates and showed no activity on any of the other aldopentoses or aldohexoses tested. With the E. coli K-12 enzyme, the K(m) was 2.8 x 10(-1)m for d-arabinose and 4.5 x 10(-2)m for l-fucose; with the E. coli B/r enzyme, the K(m) was 1.7 x 10(-1)m for d-arabinose and 4.2 x 10(-2)m for l-fucose. Both enzymes were inhibited by several of the polyalcohols tested, ribitol, l-arabitol, and dulcitol being the strongest. Both enzymes exhibited a broad plateau of optimal catalytic activity in the alkaline range. Both enzymes were stimulated by the presence of Mn(2+) or Co(2+) ions, but were strongly inhibited by the presence of Cd(2+) ions. Both enzymes were precipitated by antisera prepared against either enzyme preparation. The amino acid composition for both proteins has been determined; a striking similarity has been detected. Both enzymes could be dissociated, by protonation at pH 2 or by dialysis against buffer containing 8 m urea, into subunits that were homogeneous in an ultracentrifuge and migrated as single bands on disc electrophoresis in acrylamide gels containing urea. The molecular weight of the subunit, determined by high-speed sedimentation equilibrium, was 9.09 +/- 0.2 x 10(4) for the enzyme from E. coli K-12 and 8.46 +/- 0.1 x 10(4) for the enzyme from E. coli B/r. On the basis of biophysical studies, both isomerases appear to be oligomeric proteins consisting of four identical subunits.     

Subunit structure and higher order assembly of the hemocyanins of five members of the Naticidae family of marine gastropods: Calinaticina oldroydii (Dall), Euspira heros (Say), Neverita duplicata (Say), Polinices draconis (Dall), and Polinices lewisii (Gould)

1. The hemocyanins of the Naticidae family, E. heros, N. duplicata, P. draconis, P. lewisii and C. oldroydii were investigated by sedimentation velocity and scanning transmission electron microscopy. 2. At pH 8.0, 0.05 M Mg2+ E. heros hemocyanin is found to be predominantly in the tri-decameric state with a sedimentation coefficient (So20,w) of 131.3 (+/- 0.6) S. While the hemocyanin of N. duplicata is also mainly in the 130 S form, the hemocyanin of C. oldroydii is largely in the di-decameric form with a sedimentation coefficient close to 100 S. Other Naticidae hemocyanins, those of P. lewisii and P. draconis, have mixtures of the 100 S and 130 S di- and tri-decamers, and minor amounts of 150 S and faster sedimenting components. 3. The average particle masses based on STEM measurements are 8.85 x 10(6), 1303 x 10(6), and 17.1 x 10(6) da for the di-, tri-, and tetra-decameric assemblies of hemocyanin. 4. The subunit mol. wts of C. oldroydii hemocyanin and the published values for E. heros hemocyanin at alkaline pHs and in the presence of 8.0 M urea range from 4.2 x 10(5) to 4.8 x 10(5), suggesting the same decameric organization of the sub-assemblies of the Naticidae hemocyanins as for other molluscan hemocyanins. 5. The appearance of the larger hemocyanin particles in the electron micrographs support the hypothesis for their assembly that was based on similar studies of the hemocyanins of the Melongenidae family. According to this scheme the formation of higher aggregates is accomplished by the tail-to-head addition of each decameric unit to a central di-decamer which itself has the tail-to-tail Mellema and Klug arrangement of decamers. In this model all the higher aggregates terminate from either end with the same "collar" ends.     

Molecular weight and tertiary structure of transketolase from baker's yeast]

Molecular weight of native apotransketolase from baker's yeast is found to be 159000 +/- 6000 by means of sedimentation equilibrium and sedimentation-diffusion rate. The enzyme in a relatively low concentration reversibly dissociates into two subunits with molecular weight of about 80 000 at pH 7.6 and 20 degrees C. The equilibrium constant of the reaction monomer-dimer is 4.4 . 10(3) M-1. A decrease of the temperature stimulates the association of monomers into dimer, while the shift of pH 7.6 into acid or alkaline region stimulates the dissociation process. Dissociation becames irreversible at pH less than 5 and greater than 10.5.     

Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits.

The subunit structure of erythrocruorin from the cladoceran Daphnia magna was studied. The native protein was found to have a sedimentation coefficient (S2(20), w) of 17.9 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 494 000 +/- 33 000. Iron and haem determinations gave 0.312 +/- 0.011% and 3.84 +/- 0.04%, corresponding to minimal molecular weights of 17900 +/- 600 and 16 100 +/- 200 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a molecular weight of 31 000 +/- 1 500. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol is 31 100 +/- 1300. On a molecular-weight basis, Daphnia erythrocruorin is composed of 16 identical polypeptide chains carrying two haem groups each. The native structure is stable between pH5 and 8.5. At alkaline and acidic pH, a gradual decrease in the sedimentation coefficient down to 9.8S occurs. Above pH 10 and below pH4, a slow component with S20, w between 2.7S and 4.0S is observed. The 2.7S, 4.0S and 9.8S species are identified as single-chain subunits, subunit dimers and half-molecules respectively. We propose a model for the molecule composed of 16 2.7S subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eight-sided polygon. Support for this arrangement is provided from electron microscopy and from analysis of the pH-dissociation pattern.     

Solution studies on heme proteins: subunit structure, dissociation, and unfolding of Lumbricus terrestris hemoglobin by the ureas.

The subunit structure, dissociation, and unfolding of the hemoglobin of the earthworm, Lumbricus terrestris, were investigated by light scattering molecular weight methods and changes in optical rotatory dispersion (at 233 nm) and absorption in the Soret region. Urea and the alkylureas, methyl-, ethyl-, propyl-, and butylurea, were employed as the reagents to cause both dissociation and unfolding of the protein. Analysis of the light scattering data suggests that the dissociation patterns as a function of hemoglobin concentration in the various dissociating solvents can be described in quantitative terms, either as an equilibrium mixture consisting of parent duodecamers and hexamers of 3 x 10(6) and 1.5 x 10(6) molecular weight (in 1-3 M urea, 1-2 M methyl- and ethylurea, and 1 M propylurea), as a mixture of hexamers and monomers, the latter with a molecular weight of 250000 (i.e., in 4 M urea), or as a mixture of all three species of duodecamers, hexamers, and monomers, seen in 2 M propylurea. Parallel studies by optical rotation and absorption measurements indicate that there is little or no unfolding of the subunits at urea and alkylurea concentrations where complete dissociation to hexamers and extensive dissociation to monomers can be achieved. Further splitting of the monomers (A subunits) to smaller fragments of one-third to one-quarter of the molecular weight of the monomers (B subunits) is seen in the presence of 7 and 8 M urea (pH 7) and in alkaline urea to propylurea solutions. Analysis of the dissociation data of duodecamers to monomers, based on equations used in studies of the urea and amide dissociation of human hemoglobin A from our laboratory, suggests few urea and alkylurea binding sites at the areas of hexamer contacts in the associated duodecameric form of L. terrestris hemoglobin. This suggests that hydrophobic interactions are not the dominant forces that govern the state of association of L. terrestris hemoglobin relative to polar and ionic interactions. The unfolding effects of the ureas, at concentrations above the dissociation transitions, are closely similar to their effects on other globular proteins, suggesting that hydrophobic interactions play an important role in the maintenance of the folded conformation of the subunits. Use of the Peller-Flory equation, with binding constants based on free energy transfer data of hydrophobic amino acid side chains and denaturation data used in previous denaturation studies, gave a relatively good acount of the observed denaturation midpoints obtained with the various ureas supporting these conclusions.     

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.     

Subunit structure of the hemocyanins of some of the Muricidae and Fasciolariidae families: Chicoreus florifer dilectus (A. Adams), Muricanthus fulvescens (Sowerby), Urosalpinx cinerea (Say), Fascilaria lilium hunteria (Perry), and Pleuroploca gigantea (Kiener)

1. The hemocyanins of the Muricidae and Fasciolariidae families of marine gastropods: Chicoreus florifer dilectus, Muricanthus fulvescens, Urosalpinx cinerea, Fasciolaria lilium hunteria, and Pleuroploca gigantea were investigated by sedimentation velocity, scanning transmission electron microscopy, light-scattering, and other physical techniques. 2. The hemocyanins of these species are characterized by sedimentation coefficients close to 100 S and molecular weights of 8.2 x 10(6)-9.0 x 10(6). 3. The hemocyanins have di-decameric structures, with tail-to-tail arrangement of the decameric halves of the cylindrical particles. Only the hemocyanin of U. cinerea was found to contain about 30% higher, tri-, and tetra-decameric particles, with one or two decameric units added in a tail-to-head manner to a central di-decameric particle of the Mellema and Klug tail-to-tail arrangement. 4. The influence of pH, and the urea and Hofmeister salt series of reagents on the subunit structure and denaturation of P. gigantea hemocyanin were also investigated.