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Rat liver alcohol dehydrogenase was purified and four isoenzyme forms, demonstrated by starch gel electrophoresis, were separated by O-(carboxymethyl)-cellulose chromatography. Each of the isoenzymes had a distinct isoelectric point. All isoenzymes were active with both ethanol (or acetaldehyde) and steroid substrates, and had similar Michaelis-Menten constants for each of the substrates and coenzymes studied. The three isoenzymes with the lowest migration toward the cathode exhibited the same pH optimum of 10.7 for ethanol oxidation, a greater activity with 5 beta-androstan-3 beta-ol-17-one than with ethanol as a substrate, and an unchanged electrophoretic mobility following storage in the presence of 100 microM dithiothreitol. By contrast the isoenzyme with the highest mobility toward the cathode exhibited a pH optimum of 9.5 for ethanol oxidation, a low steroid/ethanol ratio of activity, and converted to the migrating pattern of the two isoenzymes with intermediate mobility when stored. The similarities between the isoenzymes of rat liver alcohol dehydrogenase differ considerably from differences in substrate specificity exhibited by isoenzymes of horse liver alcohol dehydrogenase.

We have characterized the size, molecular weight, and composition of the oligomeric particles produced by dialysis of bovine casein micelles against solutions lacking calcium ion. The particles were stabilized against further dissociation after dialysis by glutaraldehyde fixation. The progress of the dissociation was monitored by Biogel A-15 gel permeation chromatography, sucrose density gradient ultracentrifugation, inelastic laser light scattering, sedimentation velocity and equilibrium, and urea starch gel electrophoresis. The casein oligomers isolated after dialysis against either 0.5 m NaCl/SMUF A/azide or calcium-free SMUF/ azide have a hydrodynamic radius of about 5.5 nm and a molecular weight of about 95,000 corresponding to roughly four casein monomers (SMUF, simulated milk ultrafiltrate). The oligomers are highly hydrated and contain one-third of the calcium ion found in native micelles. During the course of dialysis, the micelles gradually break down into a broad distribution of intermediatesized particles and then into the oligomers described above.

Malate dehydrogenase (MDH) exists in several isozymic forms in the marine snail, Ilyanassa obsoleta. The isozymes may be classified in two distinct groups, mitochondrial and supernatant. The kinetic properties of these two groups of MDH's resemble those reported for mammalian and avian MDH's except that the relative electrophoretic mobilities of the two groups of isozymes are reversed. The organs of I. obsoleta all contain the same MDH isozymes as revealed by starch gel electrophoresis of organ homogenates. However, quantitative differences in relative abundance were commonly observed, but these differences may be artifacts of preparation since they were not repeatable. All of the supernatant MDH isozymes were apparently of the same molecular weight and were all convertible to a single form by prolonged exposure to 2-mercaptoethanol. This conversion was reversible by removal of the mercaptoethanol. However, the mitochondrial MDH isozymes were not affected by mercaptoethanol. Comparisons of Ilyanassa and pig heart mitochondrial and supernatant MDH isozymes showed parallel responses to mercaptoethanol by the homologous preparations from the two species.

Human scrum proteins were fractionated by electrophoresis on a “Pevikon C-870” (PVK) block. Protein fractions were recovered almost quantitatively from PVK segments and were measured for their radioactive, proteic, I131-TCA- and I131-TPA-soluble radioactivity contents. Protein fractions were characterized with respect to their electrophoretic mobilities and homogeneity on agar gel and starch gel electrophoresis and double diffusion in agar gel. Human γ-globulin, albumin, prealbumin, and orosomucoid were isolated and appeared homogeneous with the tests used in the present study.

Starch gel electrophoresis using aluminum lactate buffer (pH 3.1) containing 3 M urea demonstrated nine components in wheat gluten; four were not previously detected by the Tiselius method. One of the components appears to be high molecular weight and unable to migrate in starch gels. Gliadin was shown to be composed of eight components, whereas glutenin appeared to be identical with the high molecular weight α1 component of whole gluten. Electrophoresis of wheat gluten in Veronal (pH 7.9) and sodium glycinate (pH 9.7) buffers containing 3 M urea failed to resolve gluten into distinct components. When examined in aluminum lactate buffer containing 3 M urea, the water-soluble protein fraction isolated from a gluten preparation was composed of at least nine electrophoretic components.

A method has been developed for the purification of acid phosphomonoesterase isolated from human prostate gland. The preparation is devoid of any appreciable amount of diesterase. The method is based on preparation of a crude extract, fractionation with ammonium sulfate, extraction at pH 4.0, and chromatography and rechromatography on DEAE-cellulose. The homogeneity of the preparation was checked by means of zone electrophoresis in low-percentage agar gel with addition of Hyflo Super-Cel and by means of starch gel electrophoresis in acid and alkaline medium. In all cases the enzyme was found to be homogeneous, migrating in the form of a sharply defined band. The enzyme splits off terminal phosphate from ribonucleic acid without giving rise to its depolymerization. Spectral properties of the enzyme, relation of activity to pH, and the effect of heat denaturation are also described.

The hemoglobin types of over 150 Virginia White-tailed deer (Odocoilus Virginianus) have been studied by starch gel electrophoresis and by chromatography on columns of DEAE-Sephadex. Seventeen different types of hemoglobin heterogeneity were observed and eleven different hemoglobin components were recognized. Based on quantitative data and on subunit identification by urea starch gel electrophoresis of the globins from isolated hemoglobin components a genetic basis for the hemoglobin heterogeneity in the adult deer is proposed, which involves the existence of two non-allelic α structural genes and several allelic β structural genes.

The effect of Hg ++ and of Cr +++ on human serum was pronounced. Almost all the protein components were precipitated when serum was made 8 m M In Hg ++ or in Cr +++ . Only acid α 1 -glycoprotein was unusually stable toward Hg ++ . A serum kept in the cold for A month In The presence Of 55 m M Ag + gave a pattern with A series of aggregates of albumin. The effect of Ni ++ on human serum was less pronounced than the effect of Pb ++ , Cu ++ , and Hg ++ . The amount of γ-globulin was minimal at 10 m M of Ni ++ with The ph of the serum at 7.9. the effect of Cd ++ and Zn ++ was also mild. the serum produced larger amounts of precipitate with Cd ++ or Zn ++ at room temperature than in the cold. This behavior is occasioned by albumin which precipitates more extensively at room temperature. the effect of Ba ++ and Mg ++ was less pronounced than that of Cd ++ and Zn ++ . Only pre-albumins were precipitated at 0.1 M Ba ++ . The effect of Mgso 4 resembled that of (NH 4 ) 2 SO 4 . γ-Globulin and acid α 1 -glycoprotein were stable in a wider range of Ph and Al +++ Concentration.

Whole body and tissue homogenates of Ascaris suum exhibit four major bands of malate dehydrogenase (MDH) activity by the method of starch gel electrophoresis: one, a cathodal form which predominates in mitochondria, and the other three, anodal forms which are located primarily in the supernatant fraction of the cells. Physico-chemical studies, including thermostability, pH optima, K m 's, substrate inhibition curves, and reactivity with coenzyme analogs demonstrated differences between the mitochondrial and supernatant enzymes similar to those reported for other species. In addition the three supernatant isozymes differed in their thermostability and pH optima curves, the middle isozyme exhibiting properties intermediate to those of the other two isozymes. Observations on the ontogeny and tissue distribution of the supernatant isozymes, dissociation and recombination experiments, and the detection of an electrophoretic variant suggest that Ascaris supernatant MDH isozymes are dimers composed of subunits under separate genetic control. The functional significance of the different forms of MDH in Ascaris is discussed.

A highly purified preparation of ribulose-1,5-diphosphate carboxylase was obtained from autotrophically grown cells of Chlorella ellipsoidea . The molecular dimension of the enzyme was similar to that isolated from spinach leaf; S 20, w value of 18.4 and a cubical oligomeric structure of the protein molecule (d = ca 100 μ) as revealed by an electron microscope. Amino acid composition of the algal carboxylase was found to be similar to that of the spinach carboxylase. However, from both the quantitative immunochemical precipitin test and the enzyme-inhibition experiment using the rabbit anti-spinach RuDP carboxylase serum, there was found to exist am immunological difference between spinach and Chlorella RuDP carboxylases. The subfragment formed from the Chlorella enzyme protein by 4.0 m urea did not retain the carboxylase activity, although the spinach enzyme was split into an enzymically active subfragment under the same treatment.

Concanavalin A yielded a monodiperse pattern in the analytical ultracentrifuge in the pH range 2–5. The sedimentation coefficient, s o 20, w in pH 5 acetate buffer containing 0.1 m NaCl (total Γ 2 = 0.20 ) was 4S. At pH 7 and above, a two-peak schlieren pattern was observed, the faster component (about 7S) probably representing a dimer. On the basis of an s 20, w of 3.9S; a D 20, w of 5.43 × 10−7 cm 2 /second, and a ī V of 0.73 ml/gm, a molecular weight of 68,000 was calculated (0.10 m acetate, NaCl, Γr 2 = 0.45 ) for the homogeneous component at pH 5. From free-boundary electrophoresis data the isoelectric point of Concanavalin A was determined to be 7.1 ± 0.1. Starch gel electrophoresis patterns of concanavalin A in borate buffer (pH 8.6) were characterized by an anodically migrating streak. Incorporation of d -glucose into the gel gave a single sharp band which migrated slowly toward the cathode, the glucose probably inhibiting protein-starch interaction. Gel filtration experiments with Biogel P-100 suggested that at pH 5 the molecular weight of Concanavalin A is in the vicinity of 50,000; at pH 7.5 concanavalin A is completely excluded, an indication that a high molecular weight species probably is formed by the association of subunits. Concanavalin A has an extinction coefficient, E 1% 1 cm of 11.4 ± 0.1, the molar ratio Mtyrosine/ Mtryptophan being 1.78. Unlike other phytohemagglutinins, concanavalin A does not contain cysteine (or cystine) or carbohydrate. Aspartic acid and serine constitute the most abundant residues, and the protein has a high amide content (68.6 groups/10 5 gm protein). Amino terminal studies showed the presence of 1.5 moles DNP-alanine/ 68,000 gm protein together with very small amounts of DNP-serine and DNP-glycine. The manganese content of concanavalin A was found to be 0.029 and 0.036%, by activation analysis and emission spectroscopy, respectively.

When Pb(NO 3 ) 2 or CuSO 4 is added to human serum, the amount of precipitate increases with increase in the concentration of the cation. The supernatant was analyzed by starch-gel electrophoresis. Some protein components of human serum are precipitated at lower concentrations and some resist precipitation even at higher concentrations of a cation. The precipitability of a component depends on the nature of the cation. The main components in the supernatant obtained when serum diluted 50% is made 30 m M in Pb ++ , are transferrin and γ-globulin. Almost all the γ-globulin is precipitated when serum diluted 50% is made 6 m M in Cu ++ ; almost all the proteins are precipitated when the final concentration of Cu ++ is 20 m M . There are two interesting phenomena. One is that the zone of a particular component disappears abruptly at a certain concentration of cation, while a new zone appears, and probably indicates that the component is modified by the cation with a change in mobility. The other is that the intensity of staining of a particular component increases with increase in the cationic concentration. An explanation is that the metallic cation (Pb ++ or Cu ++ ) bound to the protein binds dye (e.g. protein − —Pb ++ —dye − ). Human serum albumin recrystallized three or four times by ammonium sulfate salting-out was analyzed by starch-gel electrophoresis. There were four zones besides the albumin zone. The pattern is similar to that obtained from human serum albumin prepared by cold ethanol fractionation. Human serum albumin extracted from the albumin zone of “starch-block” electrophoresis was shown by starch-gel electrophoresis to be more homogeneous than albumin prepared by salting-out.

Fetuin was isolated by alcohol-metal ion fractionation. Although the homogeneity of the preparation has been demonstrated by many conventional techniques, microheterogeneity was clearly demonstrated by resolution into eight components on starch gel electrophoresis at pH 4.2. The microheterogeneity did not result from the method of preparation because normal fetal calf serum and fetuin prepared by ammonium sulfate fractionation had the same electrophoretic pattern. Furthermore, the identical bands, characteristic of the purified fetuin, were present in sera collected from five fetal calves. Treatment with neuraminidase reduced the number of bands from eight to three, indicating that sialic acid is primarily responsible for the microheterogeneity observed. Fetuin which was denatured in 8 m urea or reduced with 2-mercaptoethanol in 8 m urea failed to show definite electrophoretic bands; the importance of tertiary structure as a contributory factor to the microheterogeneity is suggested. Purified fetuin was resolved into several fractions by chromatography on DEAE-Sephadex; each fraction contained 2–3 of the bands found on electrophoresis and reveals that the fetuin variants arise from discrete structural differences. The fractions showed similar immunochemical properties, sedimentation constants, and absorption coefficients at 278 mμ. The sialic acid, hexose and hexosamine content increased significantly according to the order of elution. The increase in carbohydrate was accompanied by a small decrease in peptide content. Amino acid analysis revealed virtually no relative difference between the fractions; minor differences were demonstrated by peptide mapping. It was concluded that the microheterogeneity results primarily from differences in the number and arrangement of the sialic acid residues at the surface of the molecule. Conformational variations of the polypeptide portion of the fetuin molecule may also play a contributing role.

The nuclear residual proteins of rat liver have been prepared by solubilization with sodium deoxycholate and Sephadex gel filtration. These proteins were examined by analytical ultracentrifugation, DEAE-cellulose chromatography, starch gel electrophoresis, amino acid composition, and alkali-labile phosphorus analysis. The results show that these proteins are heterogeneous, having a high glutamic acid content, and contain phosphoprotein. Partial fractionation was achieved by DEAE-cellulose chromatography and starch gel electrophoresis. Some of these proteins appeared as spheroidal particles with diameters of 100–400 A. In the presence of sodium deoxycholate, the particles dissociated into smaller units that reassociated upon removal of the detergent.

Various fractions of lactogenic hormone with different mobilities in starch gel electrophoresis have been digested with chymotrypsin and trypsin. No differences in the digestion rates were found when the enzymic reaction was followed in the pH stat by recording the alkali uptake during digestion. No significant differences were encountered when the peptide maps of the digested samples were compared. The number of bonds hydrolyzed by the enzyme as calculated from the alkali uptake during digestion was in agreement with the rate of alkali uptake during dinitrophenylation of the digested samples. No significant differences in the amounts of ether-soluble NH 2 -terminal DNP-amino acids were found in the digested and dinitrophenylated fractions. These results indicate a very close structural resemblance between the various fractions.

During purification of bovine growth hormone by Sephadex gel filtration the hormone emerged as a single asymmetrical peak from the columns. Subfractions prepared thereof proved identical by such criteria as starch gel electrophoresis, N-terminal amino acid analysis, and bioactivity. Electrophoresis of the hormone on basic and acidic polyacrylamide gels resulted in a further resolution into several well-separated bands. The ones present in sufficient amounts to be tested proved biologically and immunologically active.

Acyl phosphatase has been extracted from horse muscle and purified. The purification procedure consisted of an acid extraction, a precipitation of foreign proteins, a ZnCl 2 precipitation and two subsequent steps on Chromatographic columns of CM-Sephadex C-25. The homogeneity of the final product was established by starch gel electrophoresis, polyacrylamide gel electrophoresis, gel filtration on Sephadex G-75, and ultracentrifugation. The molecular weight was determined by the sedimentation and the Archibald procedures, the complete amino acid composition by ion exchange chromatography. Acyl phosphatase hydrolytic activity on some compounds was studied.

Two hemoglobin types have been identified by starch gel electrophoresis in stumptail macaques of both sexes and various ages. Although two hemoglobins were found in all animals, the proportion of these hemoglobins is not the same in different individuals. A comparison of the appearance and disappearance of the hemoglobins seen in the fetuses, newborns, and adults was made to the well-established ontogeny of the human hemoglobins. Structural differences that cause the electrophoretically distinguishable fetal and adult hemoglobins are described.

The availability of a pure human α 2 -macroglobulin ( α 2 -M) preparation has prompted a re-examination of its reported binding with human growth hormone (HGH). Incubation of labeled HGH preparations with α 2 -M did not show significant binding as measured by paper chromatoelectrophoresis, starch gel electrophoresis, or gel filtration. Although a small proportion of HGH traveled with α 2 -M, an analysis of the results suggests that this is not due to binding by the major component of α 2 -M. These studies indicate that the major component of α 2 -M cannot serve as a carrier protein for HGH in human plasma. An expression for the behavior of a dissociating system in gel filtration, applicable to the particular conditions employed in this study, is developed and discussed.

Both mitochondrial and cytoplasmic forms of malic dehydrogenase and aspartate aminotransferase have been demonstrated in extracts of Neurospora crassa . The mitochondrial and cytoplasmic malic dehydrogenases can readily be distinguished from each other, and from the two forms of aminotransferase, by starch gel electrophoresis. Coenzyme analogs were used to demonstrate differences in the catalytic properties of the two forms of malic dehydrogenases. Resolution of Neurospora aspartate aminotransferase and malic dehydrogenase activities was obtained by gel filtration, and the two types of activity showed differing behavior during ammonium sulfate fractionation. The present data strongly implies that in N. crassa aspartate aminotransferase and malic dehydrogenase activities reside in distinct proteins. This is in marked contrast to previous reports that both types of enzymic activity in Neurospora extracts are associated with a single mitochondrial protein.

Purified serum albumins (Fraction V) were separated into 4–5 subfractions by means of one-dimensional urea-starch gel electrophoresis in a discontinuous (Triscitrate)-borate buffer system at pH 8.6. Isolation of the three fastest migrating components of I 131 -tagged HSA and rerunning them under the same conditions gave single bands of the same mobility (as visualized by radioautography). Results with chemically modified BSA or ether-treated HSA yield patterns with fewer higher molecular weight components of decreasing mobility. No clear-cut evidence could be obtained for the presence of these albumin subfractions in normal or abnormal serum. It is therefore suggested that these multiple components constitute a series of stable higher molecular weight products or polymeric forms of an albumin monomer which result from the fractionation and purification process. Evidence is presented to support this hypothesis.

A microcolumn assay for ganglioside GM 1 β-galactosidase (EC 3.2.1.23) has been developed using GM 1 tritiated exclusively in the terminal galactose residue. The reaction is stimulated up to 100-fold by anionic and cationic detergents; this stimulation is inhibited by neutral detergents. 4-Methylumbelliferyl β- d -galactopyranoside is hydrolyzed about seven times more rapidly than GM 1 in human brain (gray matter) and liver. Agarose gel filtration separated two forms of GM 1 β-galactosidase in both brain and liver. The major form (ganglioside GM 1 β-galactosidase A) had a molecular weight of 60–70 × 10 3 and the minor form (ganglioside GM 1 β-galactosidase B) 600–800 × 10 3 . The liver and brain GM 1 β-galactosidases and 4-methylumbelliferyl β-galactosidase A cochromatographed on fractionation. The two forms of the enzyme in liver isolated by gel filtration corresponded to the two major forms found on starch gel electrophoresis and were converted to electrophoretically slower-moving forms after treatment with neuraminidase (EC 3.2.1.8, Cl. perfringens ) suggesting that both are sialylated glycoproteins. The activity of GM 1 β-galactosidase in the brain and liver tissue of patients with GM 1 gangliosidosis Types I and II was less than 2% of control values. The mutation in each GM 1 gangliosidosis appears to result in a severe reduction of activity of two ganglioside GM 1 β-galactosidases.

Reduction of the disulfide bonds of wheat gliadin and glutenin followed by electrophoresis in starch gel revealed the presence of some components which are perhaps common to both proteins. There were marked quantitative differences in the distribution of components. The release of 20 or more electrophoretic components from the previously unresolved glutenin fraction is further evidence that extensive intermolecular disulfide bonding is responsible for its high molecular weight. Intermolecular disulfide bonding is present only to a limited extent in the gliadin fraction.