Characterization of the extracellular haemoglobins of Artemia salina

The following factors were measured for extracellular haemoglobins of Artemia salina: a minimal molecular weight of globin chain per haem group (based on the iron and haem contents), the absorption coefficients, the absorption spectra of various derivatives and the amino acid compositions. These were compared with those of the haemoglobins of other invertebrates. Three Artemia haemoglobins (I, II and III) had similar molecular structures, constructed from two-globin subunits of 122000-130000mol.wt. Since the minimal mol.wt. was determined to be 18000, this suggests that one globin subunit was bound by seven haem groups, and hence one haemoglobin molecule (240000-260000mol.wt.) should contain 14 haem groups. A successful identification of this high-molecular-weight subunit required first the denaturation of haemoglobin in 1% sodium dodecyl sulphate before sodium dodecyl sulphate gel electrophoresis. Denaturation by prolonged incubation (12-36 h) at room temperature in the presence of 0.1% sodium dodecyl sulphate [Bowen, Moise, Waring & Poon (1976) Comp. Biochem. Physiol. B55, 99-103] was accompanied by extensive proteolysis, resulting in low recovery of the stainable protein and heterogeneous gel patterns. Regardless of which electrophoretic system was used, the high-molecular-weight subunit was always present provided that 1% sodium dodecyl sulphate was present during denaturation. These results contrast with those obtained by Bowen et al. (1976). However, preferential cleavage of the globin subunit (alpha) seemed to occur in vitro when standard conditions were used, producing two specific fragments having mol.wts. of 80000 (beta) and 50000 (gamma).

The purification and properties of a ribonucleoenzyme, o-diphenol oxidase, from potatoes

1. o-Diphenol oxidase was isolated from potato tubers by a new approach that avoids the browning due to autoxidation. 2. There are at least three forms of the enzyme, of different molecular weights. The major form, of highest molecular weight, was separated from the others in good yield and with high specific activity by gel filtration through Bio-Gel P-300. 3. The major form is homogeneous by disc electrophoresis but regenerates small amounts of the species of lower molecular weight, as shown by rechromatography on Bio-Gel P-300. 4. There is an equal amount of RNA and protein by weight in the fully active enzyme. The RNA cannot be removed without loss of activity, and is not attacked by ribonuclease. 5. The pH optimum of the enzyme is at pH5.0 when assayed with 4-methylcatechol as substrate. It is ten times more active with this substrate than with chlorogenic acid or catechol. The enzyme is fully active in 4m-urea. 6. A minimal molecular weight of 36000 is indicated by copper content and amino acid analysis of the protein component of the enzyme. 7. The protein contains five half-cystinyl residues per 36000 daltons, a value similar to that found in o-diphenol oxidase from mushrooms. It also contains tyrosine residues although, when pure, it does not turn brown by autoxidation.

Some Characteristics of the Clottable Protein of Limulus Polyphemus Blood Cells

Summary1. The endotoxin-clottable protein of Limulus blood cell extracts has been studied. The concept of the clottable protein as a true cell protein was confirmed.2. 35–55% of total extractable protein was removed from the extracts by clotting. Polyacrylamide disc electrophoresis of the extracts showed 6 to 9 protein bands one of which was reduced in intensity by the clotting. Dimethylf ormamide could be used for fractionated precipitation of the proteins.3. The gel protein was easily soluble in HCl or NaOH and reprecipitated by neutralization. It was also soluble in 0.05 M formate/6.7 M urea pH 4.3 but not in neutral solutions of urea.4. Light absorption spectra of the gel protein in 0.188 N NaOH showed maxima at 283 mμ and 290 mμ, whereas one maximum, at 276 mμ, was observed in 0.189 N HCl. E1 % 1 cm in 0.188 N NaOH was 10.4 and 11.1 at 283 mμ and 290 mμ, respectively, and 9.0 at 276 mμ in 0.189 N HCl.5. Data on the total amino acid composition of the gel protein are given. A mean minimal molecular weight of about 20,000 is calculated from these.6. In starch gel electrophoresis with a discontinuous acid buffer system the gel protein separated into two main zones. Possible relationships between these are discussed in terms of clotting mechanism.7. The data show that Limulus clottable protein differs markedly in its molecular characteristics from those of mammalian fibrinogens.

Characteristics of β-galactosidase in the mucosa of the small intestine of infant rats. Physicochemical properties

1. The characteristics of acid and neutral β-galactosidases isolated chromatographically from homogenates of the mucosa of the jejunum and ileum of suckling rats were studied. 2. The minimal molecular weight of the acid β-galactosidase, as estimated by gel filtration on Sephadex G-200, was in the range 83000–105000, whereas for the neutral β-galactosidase the estimated molecular weight was in the range 360000–510000. 3. The acid and neutral β-galactosidases were inhibited competitively by galactono-(1→4)-lactone, with respective Ki values of 0·15mm and 1·1mm. Only the acid β-galactosidase was inhibited competitively by sodium galactonate (Ki 0·17mm). 4. Heat inactivation of both β-galactosidases occurred according to first-order kinetics. The neutral enzyme was more labile, but bovine serum albumin protected acid enzyme only. 5. Urea treatment inactivated both β-galactosidases, the neutral β-galactosidase being more sensitive than the acid β-galactosidase. 6. No differences were found between preparations from the jejunum and ileum.

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS

1. The methods of measuring the base-combining capacities of proteins have been considered, and the constants and corrections that are employed in their calculation have been critically examined. 2. The base-combining capacities of ten casein preparations have been determined. These differed from each other to a far greater extent than can be attributed to the experimental errors involved in their measurement and calculation. The variations were, moreover, systematic in manner, and can be explained as dependent upon the method employed in the preparation of the casein. 3. Casein that had never been exposed to greater alkalinities than those in which it exists in nature combined with approximately 0.0014 mols of sodium hydroxide per gm., while casein prepared nach Hammarsten, and casein that was saturated with base during its preparation, combined with approximately 0.0018 mols of sodium hydroxide per gm. 4. 1 mol of sodium hydroxide, therefore, combined with 735 gm. of casein that had not previously been exposed to alkaline reactions, or with 535 gm. of casein that had previously been saturated with base. 5. If the minimal molecular weight of casein, based upon its tryptophane content, is placed at 12,800, the native protein must, therefore, contain approximately eighteen acid groups, and in addition six acid groups that are released in alkaline solutions, and presumably represent internally bound groups. The total base-combining capacity therefore represents that of a substance with a molecular weight of 12,800 and containing twenty-four acid valences. 6. This base-combining capacity is no greater than can be accounted for on the basis of our knowledge of the structure and composition of casein. On the basis of a molecular weight of 12,800 casein contains at least 19 molecules of glutamic acid, 4 of aspartic, and 8 of hydroxyglutamic acid. If the amino acids in the protein molecule are bound to each other in polypeptide linkage, each of these thirty-one dicarboxylic acids should yield terminal groups. The ammonia in casein suggests that twelve of these groups are bound as amides. As many as nineteen carboxyl groups may, therefore, be free in the protein molecule. 7. Casein contains phosphorus. If this phosphorus represents phosphoric acid, and if we consider that all of the valences of this acid are either themselves free, or that they have liberated carboxyl groups by entering into the structure of the protein molecule, casein should contain nine additional acid groups. 8. Recent analytical results, therefore, indicate that casein contains at least nineteen, and possibly twenty-eight, free acid groups. The physicochemical measurements presented suggest that casein combines with base as though it contained twenty-four acid groups, of which six, or one-fourth, appear to be bound in the native protein. These experimental results are therefore in close agreement with the expectation on the basis of the classical theory of protein structure.

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS

1. The solubility in water of purified, uncombined casein has previously been reported to be 0.11 gm. in 1 liter at 25°C. This solubility represents the sum of the concentrations of the casein molecule and of the soluble ions into which it dissociates. 2. The solubility of casein has now been studied in systems containing the protein and varying amounts of sodium hydroxide. It was found that casein forms a well defined soluble disodium compound, and that solubility was completely determined by (a) the solubility of the casein molecule, and (b) the concentration of the disodium casein compound. 3. In our experiments each mol of sodium hydroxide combined with approximately 2,100 gm. of casein. 4. The equivalent combining weight of casein for this base is just half the minimal molecular weight as calculated from the sulfur and phosphorus content, and one-sixth the minimal molecular weight calculated from the tryptophane content of casein. 5. From the study of systems containing the protein and very small amounts of sodium hydroxide it was possible to determine the solubility of the casein molecule, and also the degree to which it dissociated as a divalent acid and combined with base. 6. Solubility in such systems increased in direct proportion to the amount of sodium hydroxide they contained. 7. The concentration of the soluble casein compound varied inversely as the square of the hydrogen ion concentration, directly as the solubility of the casein molecule, Su, and as the constants Ka1 and Ka2 defining its acid dissociation. 8. The product of the solubility of the casein molecule and its acid dissociation constants yields the solubility product constant, Su·Ka1·Ka2 = 2.2 x 10–12 gm. casein per liter at 25°C. 9. The solubility of the casein molecule has been estimated from this constant, and also from the relation between the solubility of the casein and the sodium hydroxide concentration, to be approximately 0.09 gm. per liter at 25°C. 10. The product of the acid dissociation constants, Ka1 and Ka2, must therefore be 24 x 10–12N. 11. It is believed that these constants completely characterize the solubility of casein in systems containing the protein and small amounts of sodium hydroxide.