A PHASE RULE STUDY OF THE PROTEINS OF BLOOD SERUM: THE EFFECT OF CHANGES IN CERTAIN VARIABLES

Changes were studied in the standard solubility curve of fresh serum proteins by alterations in pH, temperature, concentration of protein, and nature of the salt used for precipitation. The principal factor affecting the precipitation of protein fractions was a change in temperature. In order to investigate the proteins in their original states low temperatures are necessary. Protein fraction A is altered by a change in pH and with the use of (NH₄)₂SO₄ as a precipitant, fraction B by a change in pH and temperature, and use of (NH₄)₂SO₄, C by a change in temperature and concentration of the protein, and D by a change in temperature and pH. The solubility of D is independent of the amount of protein in solution in high concentrations of salt.     

A PHASE RULE STUDY OF THE PROTEINS OF BLOOD SERUM : III. GLOBULIN

A method has been developed for applying the phase rule to systems of several protein components in serum. The globulin fractions which have been investigated appear to be homogeneous substances.     

DISTRIBUTION OF SERUM PROTEINS AND BETA-TRACE PROTEIN WITHIN THE NERVOUS SYSTEM

Abstract— The concentration of beta-trace protein, a low molecular weight water-soluble protein, was significantly higher in cerebral and cerebellar white matter than in grey matter. A similar distribution was found for transferrin. The concentrations of gamma-trace protein and pre-albumin were almost constant in cerebral and cerebellar white and grey matter. A different distribution was shown for albumin, beta_lc/beta_lA globulins, and the immunoglobulins G, A and M, with the highest concentrations mostly encountered in the highly vascularized basal ganglia and grey matter, and the lowest concentrations in white matter. Thus, different parameters, hitherto unknown determine the distribution within the central nervous system of various proteins-those which originate from serum, and beta-trace protein which originates mainly from the central nervous system.
The amounts of the different proteins were higher in the choroid plexus than in brain tissue, with the exception of gamma-trace protein.
Foetal brains contained increasing concentrations of beta-trace protein and of transferrin with age.
Femoral nerve contained lower concentrations of beta-trace protein and gamma-trace protein, and higher concentrations of the other proteins, than the central nervous system.     

THE USE OF SOLUBILITY AS A CRITERION OF PURITY OF PROTEINS : I. APPLICATION OF THE PHASE RULE TO THE SOLUBILITY OF PROTEINS. II. THE SOLUBILITY CURVES AND PURITY OF CHYMOTRYPSINOGEN

1. The conditions under which the phase rule may be applied to systems containing proteins are formulated. 2. An attempt was made to fractionate chymotrypsinogen, by crystallization in stages with increasing concentration of magnesium sulfate. No significant fractionation of the protein was achieved, but a small amount of impurity which affects the solubility, while having little influence on other properties of the material, was concentrated in the fractions first precipitated. 3. The solubility of the final fraction was independent of the amount of the saturating solid, from the first appearance of a solid phase, in solvents of three different pH''s. The solubility was independent of the environment in which the crystals were formed (within the limits in which crystallization can be carried out) and the same value was reached from the supersaturated as from the undersaturated side. This material, therefore, conforms closely with the phase rule criteria of a pure protein.     

THE REDUCING GROUPS OF PROTEINS

1. Intact, unhydrolyzed proteins possess in addition to SH groups other reducing groups which can be oxidized by ferricyanide. 2. The activity of these reducing groups, like that of SH groups, is enhanced by denaturation of the protein and by increase of pH and temperature. 3. These groups differ from SH groups in the manner in which their activity is dependent on concentration of ferricyanide and time of contact with ferricyanide. 4. The activity of these groups is increased if protein SH groups are present. 5. The number and activity of these groups varies from protein to protein. 6. These groups are probably contained in the tyrosine and tryptophane components of proteins. 7. The significance of these reducing groups for an understanding of protein denaturation and the reducing properties of tissues is indicated.     

PROTEINS OF THE POSTSYNAPTIC DENSITY

An analysis was made of the protein composition of a fraction of postsynaptic densities (PSDs) prepared from rat brain. Protein makes up 90% of the material in the PSD fraction. Two major polypeptide fractions are present, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The major polypeptide fraction has a molecular weight of 53,000, makes up about 45% of the PSD protein, and comigrates on gels with a major polypeptide of the synaptic plasma membrane. The other polypeptide band has a molecular weight of 97,000, accounts for 17% of the PSD protein, and is not a prominent constituent of other fractions. Six other polypeptides of higher molecular weight (100,000–180,000) are consistently present in small amounts (3–9% each). The PSD fraction contains slightly greater amounts of polar amino acids and proline than the synaptic plasma membrane fraction, but no amino acid is usually prominent. The PSD apparently consists of a structural matrix formed primarily by a single polypeptide or class of polypeptides of 53,000 molecular weight. Small amounts of other specialized proteins are contained within this matrix.     

THE STABILITY OF BACTERIAL SUSPENSIONS : III. AGGLUTINATION IN THE PRESENCE OF PROTEINS,NORMAL SERUM,AND IMMUNE SERUM.

1. The addition of proteins or serum to suspensions of bacteria, (Bacillus typhosus or rabbit septicemia) at different pH widens the acid agglutination zone and shifts the isoelectric point to that of the added substance. 2. The amount of serum required to agglutinate is much less near the acid agglutination point of the organisms. 3. The addition of immune serum prevents the salt from decreasing the cohesive force between the organisms, and agglutination therefore is determined solely by the potential, provided excess immune body is present. Whenever the potential is decreased below 15 millivolts the suspension agglutinates.     

黄瓜器官特异蛋白的研究

利用 SDS单向电泳对黄瓜 ( Cucumissativus L .)的根、茎、叶、花萼、花冠、雄蕊、花柱和子房的可溶性蛋白进行了分析和比较。检测到花冠中的 2 3.5 k D和 33.0 k D,雄蕊中的 1 8.8k D、2 8.5 k D、31 .0 k D、37.0 k D和 39.0 k D,花柱中的 4 5 .0 k D及子房中的 32 .5 k D蛋白 ,分别为各自器官中的器官特异蛋白质。对花冠、雄蕊、花柱和子房的可溶性蛋白的 IEF- SDS双向电泳分析也确定了相应于 SDS单向电泳上特异蛋白带的蛋白质斑点。而且相应于 SDS单向电泳上的一条带 ,在 IEF- SDS双向电泳上可能是一个以上的分子量相同而等电点不同的几个蛋白质斑点。各种器官的蛋白质含量以雄蕊为最高、花萼为最低。     

THE SIGNIFICANCE OF THE HYDROGEN ION CONCENTRATION FOR THE DIGESTION OF PROTEINS BY PEPSIN

The experiments described above show that the rate of digestion and the conductivity of protein solutions are very closely parallel. If the isoelectric point of a protein is at a lower hydrogen ion concentration than that of another, the conductivity and also the rate of digestion of the first protein extends further to the alkaline side. The optimum hydrogen ion concentration for the rate of digestion and the degree of ionization (conductivity) of gelatin solutions is the same, and the curves for the ionization and rate of digestion as plotted against the pH are nearly parallel throughout. The addition of a salt with the same anion as the acid to a solution of protein already containing the optimum amount of the acid has the same depressing effect on the digestion as has the addition of the equivalent amount of acid. These facts are in quantitative agreement with the hypothesis that the determining factor in the digestion of proteins by pepsin is the amount of ionized protein present in the solution. It was shown in a previous paper that this would also account for the peculiar relation between the rate of digestion and the concentration of protein. The amount of ionized protein in the solution depends on the amount of salt formed between the protein (a weak base) and the acid. This quantity, in turn, according to the hydrolysis theory of the salts of weak bases and strong acids, is a function of the hydrogen ion concentration, up to the point at which all the protein is combined with the acid as a salt. This point is the optimum hydrogen ion concentration for digestion, since the solution now contains the maximum concentration of protein ions. The hydrogen ion concentration in this range therefore is merely a convenient indicator of the amount of ionized protein present in the solution and takes no active part in the hydrolysis. After sufficient acid has been added to combine with all the protein, i.e. at pH of about 2.0, the further addition of acid serves to depress the ionization of the protein salt by increasing the concentration of the common anion. The hydrogen ion concentration is, therefore, no longer an indicator of the amount of ionized protein present, since this quantity is now determined by the anion concentration. Hence on the acid side of the optimum the addition of the same concentration of anion should have the same influence on the rate of digestion irrespective of whether it is combined with hydrogen or some other ion (provided, of course, that there is no other secondary effect of the other ion). The proposed mechanism is very similar to that suggested by Stieglitz and his coworkers for the hydrolysis of the imido esters. Pekelharing and Ringer have shown that pure pepsin in acid solution is always negatively charged; i.e., it is an anion. The experiments described above show further that it behaves just as would be expected of any anion in the presence of a salt containing the protein ion as the cation and as has been shown by Loeb to be the case with inorganic anions. Nothing has been said in regard to the quantitative agreement between the increasing amounts of ionized protein found in the solution (as shown by the conductivity values) and the amount predicted by the hydrolysis theory of the formation of salts of weak bases and strong acids. There is little doubt that the values are in qualitative agreement with such a theory. In order to make a quantitative comparison, however, it would be necessary to know the ionization constant of the protein and of the protein salt and also the number of hydroxyl (or amino) groups in the protein molecule as well as the molecular weight of the protein. Since these values are not known with any degree of certainty there appears to be no value at present in attempting to apply the hydrolysis equations to the data obtained. It it clear that the hypothesis as outlined above for the hydrolysis of proteins by pepsin cannot be extended directly to enzymes in general, since in many cases the substrate is not known to exist in an ionized condition at all. It is possible, however, that ionization is really present or that the equilibrium instead of being ionic is between two tautomeric forms of the substrate, only one of which is attacked by the enzyme. Furthermore, it is clear that even in the case of proteins there are difficulties in the way since the pepsin obtained from young animals, or a similar enzyme preparation from yeast or other microorganisms, is said to have a different optimum hydrogen ion concentration than that found for the pepsin used in these experiments. The activity of these enzyme preparations therefore would not be found to depend on the ionization of the protein. It is possible of course that the enzyme preparations mentioned may contain several proteolytic enzymes and that the action observed is a combination of the action of several enzymes. Dernby has shown that this is a very probable explanation of the action of the autolytic enzymes. The optimum hydrogen ion concentration for the activity of the pepsin used in these experiments agrees very closely with that found by Ringer for pepsin prepared by him directly from gastric juice and very carefully purified. Ringer''s pepsin probably represents as pure an enzyme preparation as it is possible to prepare. There is every reason to suppose therefore that the enzyme used in this work was not a mixture of several enzymes.     

THE EFFECT OF VARIOUS ACIDS ON THE DIGESTION OF PROTEINS BY PEPSIN

1. At equal hydrogen ion concentration the rate of pepsin digestion of gelatin, egg albumin, blood albumin, casein, and edestin is the same in solutions of hydrochloric, nitric, sulfuric, oxalic, citric, and phosphoric acids. Acetic acid diminishes the rate of digestion of all the proteins except gelatin. 2. There is no evidence of antagonistic salt action in the effect of acids on the pepsin digestion of proteins. 3. The state of aggregation of the protein, i.e. whether in solution or not, and the viscosity of the solution have no marked influence on the rate of digestion of the protein.     

THE BLOCKAGE OF AXOPLASMIC FLOW OF PROTEINS BY COLCHICINE AND CYTOCHALASINS A AND B

Abstract— Colchichine blocks axoplasmic flow of proteins in chicken sciatic nerve. The slow component is more effectively blocked than the fast. The colchicine effect on slow flow is independent of the time delay between colchicine injection and that of the [¹⁴C]-leucine used to measure flow, over a period extending from 2 h after to 9 days before the leucine. It is still effective, but to a lesser degree, after a period of 27 days. There is little effect on the fast component when the colchicine is administered after leucine. When given before leucine the effect is not pronounced up to a time interval of 1 day. Maximum blockage was obtained with longer intervals of up to 27 days. These results are discussed in relation to the involvement of the microtubules in both slow flow and rapid flow.
The effect of both cytochalasins A and B on slow and rapid protein flow has also been studied. Neither drug had any significant effect on slow flow. Cytochalasin A reduced the amount of protein flowing at the rapid rate to a small extent when administered 2 h before [¹⁴C]leucine. Cytochalasin B also caused a similar reduction and this effect was independent of dose over the ranges of doses used. The cytochalasin B diminished the incorporation of amino acid into protein in the spinal cord and it has been concluded that these results are due to a membrane effect which reduces uptake of amino acid rather than a direct effect on neurofilaments in the axons.     

THE FLOCCULATION OF BACTERIA BY PROTEINS

1. The effect of adding pure proteins to bacterial suspensions at different H ion concentrations has been studied. 2. The zone of flocculation of protein-treated bacteria bears a significant relationship to the isoelectric point of the protein used. With the higher concentration of protein, agglutination occurs at or near the isoelectric point of that protein; at reactions acid to this, the bacteria carry a positive charge and are not agglutinated. With diminishing concentration of protein, the zone of flocculation shifts toward and goes beyond that characteristic of the untreated bacteria. This occurs both in the presence and absence of salts. 3. A diversity of other suspensions, such as sols of gold, mastic, cellulose nitrate, cellulose acetate, Fe(OH)₃, oil emulsions, and erythrocytes, have been found by ourselves and others to exhibit a similar altered stability when treated with proteins in the same way.     

兰州百合精细胞特异蛋白的研究

通过低渗冲击及Percoll密度梯度离心的方法,成功地分离并纯化了兰州百合(Lilium davidiiDuch.)生活的生殖细胞及精细胞。从精细胞、生殖细胞及叶片中提取了全蛋白,并通过双向电泳技术对它们进行了比较。在双向电泳图谱上精细胞比生殖细胞显示更多的蛋白斑点,特别是在碱性端。通过混合酶解及离心,分离了生活的叶肉原生质体。用生物素的琥珀酰胺酯衍生物(NHS-biotin)对精细胞、生殖细胞及完整的叶肉原生质体质膜蛋白进行标记,然后进行Western blot分析,用辣根过氧化物酶酶标链霉抗生物素蛋白及其底物4-氯-1-萘酚反应显色,比较了3种质膜蛋白。发现分子量为46kD及50kD的两种蛋白是精细胞质膜特异的。在双向电泳图谱上也可找到与这两种蛋白相对应的斑点,它们很可能与受精过程中精卵的识别有关。     

REGIONAL AND SEXUAL DISTRIBUTION PATTERNS OF CHROMATIN PROTEINS AND OF SOLUBLE CYTOPLASMIC PROTEINS IN THE RAT BRAIN1

Abstract— Electrophoretic patterns of forebrain and hindbrain proteins were compared in normal adult rats of both sexes, as well as in adult female rats following neonatal androgenization. There were significant differences between the forebrains and hindbrains in the relative densities of several chromatin proteins and soluble cytoplasmic protein bands. Minor qualitative differences between forebrains and hindbrains were observed in patterns of chromatin proteins. No qualitative sex difference was detectable in the patterns of chromatin-derived proteins or in soluble cytoplasmic proteins. The relative densities of the brain protein bands were very similar for both sexes, indicating quantitative equivalence as well. The brain protein electrophoretic patterns of adult females following neonatal androgenization were identical qualitatively to those of normal females in respect to both chromatin and cytoplasmic proteins.     

MYELIN PROTEINS FROM DIFFERENT REGIONS OF THE CENTRAL NERVOUS SYSTEM

—The protein composition of myelin prepared from specific anatomical regions of the bovine brain and spinal cord was studied by a modification of the method of Gonzalez -Sastre (1970). Spinal cord myelin contained lesser amounts of chloroform-methanol soluble protein and proteolipid protein and had a lower activity of the enzyme 2′,3′-cyclic nucleotide 3′-phosphohydrolase than did myelin from subcortical white matter. There was no difference, however, in the protein composition of myelin from the various levels of the spinal cord. The amino acid composition of both proteolipid and basic protein showed no significant regional differences. Myelin preparations from both brain and spinal cord contained DM-20 protein.     

ELECTROKINETIC PHENOMENA : I. THE ADSORPTION OF SERUM PROTEINS BY QUARTZ AND PARAFFIN OIL

1. The effect of human and rabbit sera on the cataphoretic mobility of glass and quartz particles, and of paraffin oil droplets, was studied in serum dilutions (with 0.85 per cent NaCl) from 1:50 to 1:1,000,000, over a pH range of 3.6 to 9.3. 2. Under the conditions described, these various types of particles adsorbed protein partially or completely from the most dilute solution giving these particles electrokinetic properties characteristic of certain proteins, probably here those of serum albumin. 3. Quartz particles and paraffin oil droplets both have an isoelectric point between 4.7 and 4.8 in a 1:50 serum dilution. 4. The biological importance of these findings is discussed. 5. A non-polarizable electrode composed of See PDF for Structure is described for use with cataphoresis cells.     

THE DETERMINATION OF THE EQUIVALENT WEIGHT OF PROTEINS

The magnitude of the correction in the fifth column of Table III may be open to some doubt, as are all corrections of such a character, and the significance of the above experiment in the author''s mind lies not so much in the actual magnitude of the values given in the last column of this table as in their comparative magnitudes. For this reason the entire experiment reported was performed in a single session using the same gelatin solution, so that, whatever the magnitude of the correction, it would be the same in all cases. Actually the results in the case of the acid titrations are in fair agreement with those of Hitchcock (8). In the present experiment it is seen that, within the limits of experimental error, one gets the same value for the number of cc. of tenth normal acid bound by 1 gm. of gelatin whether one titrates with the acid or with the gelatin. In the case of the base there is a small difference, due probably to carbon dioxide, but this effect is in a direction opposite to that which one would expect on the assumption that it is due to appreciable adsorption. From this it is concluded that the binding due to adsorption in the case of gelatin is not significant compared to that due to chemical neutralization. The author realizes that gelatin is a poor choice for a basis of generalizations, and similar work is at present in progress on various other proteins. He does feel, however, that the conclusions of Hoffman and Gortner from their work on the prolamines may also be too widely generalized, and that, on the whole, the acid or alkali bound by adsorption in the case of proteins will not constitute the large majority of the total amounts bound, though certainly one will expect a certain amount of such binding in all cases. It also seems that before placing undue emphasis on the conclusions of these workers the possibilities of equivocal results due to specific technique should be considered. This technique consisted in introducing weighed amounts of dry protein into a definite volume of standard acid or base at the equilibrium temperature, in general, and, "after about 15 minutes, during which time the flask was shaken several times," determining the pH of the equilibrium solution. Is it possible that the actual speed of solution of the protean is such that, even though reproducible results are obtained using identical technique, actual equilibrium conditions are approached only when comparatively high concentrations of acid or alkali are employed, in which cases the solution velocity of the protein may he expected to be greater, other factors remaining constant?     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : I. THE SOLUBILITY OF CERTAIN PROTEINS AT THEIR ISOELECTRIC POINTS.

1. Two proteins of the globulin type, serum globulin and tuberin, and the protein of milk, casein, have been purified (a) of the other proteins and (b) of the inorganic electrolytes with which they exist in nature. The methods that were employed are described. 2. All three proteins were found to be only very slightly soluble in water in the pure uncombined state. The solubility of each was accurately measured at 25.0° ± 0.1°C. The most probable solubility of the pseudoglobulin of serum was found to be 0.07 gm. in 1 liter; of tuberin 0.1 gm. and of casein 0.11 gm. The methods that were employed in their determination are described. 3. Each protein investigated dissolved in water to a constant and characteristic extent when the amount of protein precipitate with which the solution was in heterogeneous equilibrium was varied within wide limits. The solubility of a pure protein is therefore proposed as a fundamental physicochemical constant, which may be used in identifying and in classifying proteins. 4. The concentration of protein dissolved must be the sum of the concentration of the undissociated protein molecule which is in heterogeneous equilibrium with the protein precipitate, and of the concentration of the dissociated protein ions. 5. The dissociated ions of the dissolved protein give a hydrogen ion concentration to water that is also a characteristic of each protein.