PROTEIN COAGULATION AND ITS REVERSAL : THE IDENTITY OF NORMAL HEMOGLOBIN WITH THE HEMOGLOBIN PREPARED BY THE REVERSAL OF COAGULATION,AS DETERMINED BY SOLUBILITY TESTS

1. Methemoglobin prepared from coagulated hemoglobin by the reversal of coagulation has the same solubility within 2 per cent as normal methemoglobin. 2. Methemoglobin synthesized from hemin and the native globin prepared by the reversal of coagulation of globin likewise has the same solubility as normal methemoglobin.     

PROTEIN COAGULATION AND ITS REVERSAL : GLOBIN

1. The globin prepared from hemoglobin by the acid acetone method is denatured globin. 2. The denaturation and coagulation of globin by acid acetone are reversible. 3. Soluble globin can be obtained from the acid acetone globin even if the globin is first precipitated by trichloracetic acid or heated to 100°C. 4. Hill and Holden''s theory that they separated native globin from hemoglobin without any intermediate denaturation is not proven by their experiments.     

PROTEIN COAGULATION AND ITS REVERSAL : SERUM ALBUMIN

1. It is possible to prepare crystalline, soluble, heat-coagulable serum albumin from coagulated serum albumin. 2. In the cases so far studied, the more soluble a denatured protein, the more easily its denaturation can be reversed.     

COAGULATION OF MYOSIN BY DEHYDRATION

When myosin is dehydrated it becomes insoluble. The number of detectable SH groups in myosin coagulated by dehydration is the same as in native soluble myosin. In this respect coagulation by dehydration differs from coagulation brought about in any of the other ways now known, but resembles the coagulation that occurs in muscle during rigor and in the egg after fertilization.     

THE PREPARATION OF COMPLETELY COAGULATED HEMOGLOBIN

As a preliminary to the study of the reversal of the coagulation of hemoglobin several methods are described for the preparation of completely denatured and coagulated hemoglobin and the evidence is given that hemoglobin is a typical coagulable protein.     

THE COAGULATION OF MYOSIN IN MUSCLE

1. Muscle can be prepared in the form of a dry powder in which myosin exists in a state similar to that in intact muscle. As in intact muscle, myosin in powdered muscle is soluble and can be caused to rapidly coagulate. 2. Restoring to powdered muscle the quantity of water previously removed causes coagulation of myosin. The rate of coagulation is considerably slower at 0° than at 20°. 3. Adding the powder to a large volume of dilute salt solution also results in coagulation. 4. The water soluble constituents of muscle can be removed from the powder without thereby causing coagulation. Coagulation occurs in water extracted muscle when it is suspended in a dilute salt solution. 5. Coagulation of myosin in muscle resembles the coagulation of myosin caused by dehydration. 6. Myosin coagulates readily only when it is imbedded in the structure of muscle. The significance for coagulation of the arrangement of myosin particles in muscle has been indicated.     

SULFHYDRYL AND DISULFIDE GROUPS OF PROTEINS : II. THE RELATION BETWEEN NUMBER OFSH AND S-S GROUPS AND QUANTITY OF INSOLUBLE PROTEIN IN DENATURATION AND IN REVERSAL OF DENATURATION

1. In native egg albumin no SH groups are detectable, whereas in completely coagulated albumin as many groups are detectable as are found in the hydrolyzed protein. In egg albumin partially coagulated by heat the soluble fraction contains no detectable groups, and the insoluble fraction contains the number found after hydrolysis. 2. In the reversal of denaturation of serum albumin, when insoluble protein regains its solubility, S-S groups which have been detectable in the denatured protein, disappear. 3. When egg albumin coagulates at an air-water interface, all the SH groups in the molecule become detectable. 4. In egg albumin coagulated by irradiation with ultraviolet light, the same number of SH groups are detectable as in albumin coagulated by a typical denaturing agent. 5. When serum albumin is denatured by urea, there is no evidence that S-S groups appear before the protein loses its solubility. 6. Protein denaturation is a definite chemical reaction: different quantitative methods agree in estimates of the extent of denaturation, and the same changes are observed in the protein when it is denatured by different agents. A protein molecule is either native or denatured. The denaturation of some proteins can be reversed.     

DISTRIBUTION OF COAGULATION OF YOUNG SHOOT HOMOGENATES IN THE AURANTIOIDEAE

Distribution of coagulation of young shoot homogenates was surveyed in 430 accessions from the orange subfamily Aurantioideae. Representatives of four genera available for study (Clausena, Glycosmis, Aegle, Feronia) were coagulating taxa. The genus Citrus included both coagulating and noncoagulating taxa. Nineteen genera (Murraya, Merillia, Pamburus, Paramignya, Triphasia, Aeglopsis, Afraegle, Balsamocitrus, Feroniella, Swinglea, Atalantia, Citropsis, Hesperethusa, Pleiospermium, Severinia, Clymenia, Eremocitrus, Poncirus, Fortunella) were noncoagulating. The status of eight genera (Micromelum, Wenzelia, Monanthocitrus, Oxanthera, Merope, Luvunga, Burkillanthus, Limnocitrus) is not known. Homogenized tissue from coagulating taxa turned into a paste while tissue from noncoagulating taxa was thin and juicy. Coagulation did not take place when the pH of the grinding medium was 11 or higher but it would occur if the pH was lowered. Homogenates of noncoagulating taxa become coagulated below pH 5.2–5.3, but remained noncoagulating above this. Noncoagulating taxa contained an unidentified substance which inhibited coagulation of tissue from coagulating taxa. The mechanism of coagulation and noncoagulaton is not presently known. The significance of coagulation and its absence as a taxonomic and genetic marker is discussed.     

外源途径凝血的抑制作用的模型与模拟(一)-蛋白C的动力学影响

振荡与周期性节律已经在生物医学的众多领域及各层次的研究中出现,此文首次建立了一个关于蛋白Ｃ的凝血动力系统模型。通过对模型的动力学分析得到了一些新的结论,这些结论将对血液学理论与临床血液学提供有益的启示,特别是周期性振荡的终态存在性是十分有趣的现象,另外还得到了一些与临床医学及实验相吻和的定性结论,展现出模型化方法研究凝血系统动力学的重要作用及预测性能力     

THE INFLUENCE OF DETERGENTS ON SOME PHYSIOLOGICAL PHENOMENA,ESPECIALLY ON THE PROPERTIES OF THE STELLATE CELLS OF THE FROG LIVER

1. After a consideration of the physicochemical properties of detergents, it was deemed worth while to study some of their physiological effects. As nonpolar-polar electrolytes, the detergents are surface-active and as such cytolytics; but probably due to their dispersing and wetting properties, they are cytolytic in a fashion different from that of other cytolytics. The detergents tested were alkyl sulfonates, alkyl sulfosuccinates, and bile salts. 2. The cytolytic power has been tested in two ways, (1) with red cells by following the escape of hemoglobin, (2) with muscles by measuring the development of an injury potential. In both series of experiments the threshold concentrations of action have been determined. The effect on the potentials has proved to be, in general, reversible. 3. The hemolytic and the myolytic power run fairly parallel to the surface activity. 4. Dehydrocholate has been found to be lacking in nonpolar-polar properties. 5. The stellate cells (Kupffer cells) of the Ringer-perfused frog liver are unable to take up colloidal dyestuffs (trypan blue and soluble blue R), except after addition of a small amount of serum to the perfusing Ringer solution. Only under the latter conditions, the uptake of dye is increased by adding a detergent. This seems to be due to the combined action of the proteins and the detergents. 6. The effect of relatively high concentrations of detergent is disintegration of the stellate cells; viz., cytolysis. There are reasons to assume that small concentrations, which produce a threshold increase of the dyestuff uptake, raise the functional activity.     

ON SOME GENERAL PROPERTIES OF PROTEINS

1. The processes of denaturation and coagulation of hemoglobin are like those of other proteins. 2. When hemoglobin is denatured it is probably depolymerized into hemochromogen. 3. When other proteins are denatured they, too, are probably depolymerized. Conversely, native proteins can be regarded as aggregates of denatured proteins. 4. The globins and histones are to be regarded as denatured proteins rather than as a distinct group of proteins. 5. The factors affecting the equilibrium between native and denatured proteins have been considered. 6. A non-polar group is uncovered when a protein is denatured. 7. It has been shown that judged by the two most sensitive tests for the specificity of proteins, it is only when proteins are in the native form that they are highly specific.     

A TEST FOR DIFFUSIBLE IONS : II. THE IONIC NATURE OF PEPSIN.

1. The distribution of pepsin between particles of gelatin or coagulated egg albumin and the outside solution has been found to be equal to the distribution of chloride or bromide ion under the same conditions. This is the case from pH 1 to 7, and in the presence of a variety of salts. 2. Pepsin is therefore probably a monovalent anion. 3. Under certain conditions the enzyme may be adsorbed on the surface of the protein particles. This reaction is irreversible and is markedly influenced by the presence of low concentrations of electrolytes.     

THE REVERSAL OF THE SIGN OF THE CHARGE OF MEMBRANES BY HYDROGEN IONS

1. It had been shown in previous papers that when a collodion membrane has been treated with a protein the membrane assumes a positive charge when the hydrogen ion concentration of the solution with which it is in contact exceeds a certain limit. It is pointed out in this paper that by treating the collodion membrane with a protein (e.g. oxyhemoglobin) a thin film of protein adheres to the membrane and that the positive charge of the membrane must therefore be localized in this protein film. 2. It is further shown in this paper that the hydrogen ion concentration, at which the reversal in the sign of the charge of a collodion membrane treated with a protein occurs, varies in the same sense as the isoelectric point of the protein, with which the membrane has been treated, and is always slightly higher than that of the isoelectric point of the protein used. 3. The critical hydrogen ion concentration required for the reversal seems to be, therefore, that concentration where enough of the protein lining of the membrane is converted into a protein-acid salt (e.g. gelatin nitrate) capable of ionizing into a positive protein ion (e.g. gelatin) and the anion of the acid used (e.g. NO₃).     

IONIZING INFLUENCE OF SALTS WITH TRIVALENT AND TETRAVALENT IONS ON CRYSTALLINE EGG ALBUMIN AT THE ISOELECTRIC POINT

1. While crystalline egg albumin is highly soluble in water at low temperature at the pH of its isoelectric point, it is coagulated by heating. It has long been known that this coagulation can be prevented by adding either acid or alkali, whereby the protein is ionized. 2. It is shown in this paper that salts with trivalent or tetravalent ions, e.g. LaCl₃ or Na₄Fe(CN)₆, are also able to prevent the heat coagulation of albumin at the isoelectric point (i.e. pH 4.8), while salts with a divalent ion, e.g. CaCl₂, BaCl₄, Na₂SO₄, or salts like NaCl, have no such effect. 3. This is in harmony with the fact shown in a preceding paper that salts with trivalent or tetravalent ions can cause the ionization of proteins at its isoelectric point and thus give rise to a membrane potential between micellæ of isoelectric protein and surrounding aqueous solution, while the above mentioned salts with divalent and monovalent ions have apparently no such effect.     

FRACTIONATION OF PEPSIN : I. ISOLATION OF CRYSTALLINE PEPSIN OF CONSTANT ACTIVITY AND SOLUBILITY FROM PEPSINOGEN OR COMMERCIAL PEPSIN PREPARATIONS. II. PREPARATION OF A LESS SOLUBLE FRACTION. III. SOLUBILITY CURVES OF MIXTURES OF THE SOLUBLE AND INSOLUBLE FRACTIONS. IV. PREPARATION OF HIGHLY ACTIVE PEPSIN FROM PEPSINOGEN

1. Solubility curves of crude pepsin preparations indicate the presence of more than one protein. 2. One of these proteins has been isolated and crystallized and found to have constant activity and constant solubility in several solvents. 3. The solubility measurements are complicated by the unstable nature of the protein and the fact that in certain solvents the solubility of the protein is markedly affected by the presence of non-protein nitrogen decomposition products while in others this is not the case. 4. A more insoluble protein has been prepared of lower solubility and lower activity, as measured by hemoglobin digestion. The activity, as measured by the digestion of other proteins, is about the same as the more soluble fraction. This insoluble fraction does not have constant solubility. 5. Mixtures of the insoluble and the soluble fractions give preparations having rounded solubility curves typical of solid solutions and resembling very closely those of the original preparation. 6. A small amount of pepsinogen and pepsin from pepsinogen has been separated which has nearly twice the enzymatic activity on hemoglobin as does the most active pepsin previously isolated.     

THE MECHANISM OF THE INFLUENCE OF ACIDS AND ALKALIES ON THE DIGESTION OF PROTEINS BY PEPSIN OR TRYPSIN

1. The effect of the addition of acid on the amount of ionized protein has been compared with the effect on the rate of digestion of gelatin, casein, and hemoglobin by pepsin. 2. A similar comparison has been made of the addition of alkali in the case of trypsin with gelatin, casein, hemoglobin, globin, and edestin. 3. In general, the rate of digestion may be predicted from the amount of ionized protein as determined by the titration curve or conductivity. The rate of digestion is a minimum at the isoelectric point of the protein and a maximum at that pH at which the protein is completely combined with acid or alkali to form a salt. 4. The physical properties of the protein solution have little or no effect on the rate of digestion.     

THE EQUILIBRIA BETWEEN NATIVE AND DENATURED HEMOGLOBIN IN SALICYLATE SOLUTIONS AND THE THEORETICAL CONSEQUENCES OF THE EQUILIBRIUM BETWEEN NATIVE AND DENATURED PROTEIN

The denaturation of hemoglobin by salicylate in neutral solution is completely reversible. There is a mobile equilibrium between native and denatured hemoglobin in neutral salicylate solution. The higher the salicylate concentration the greater is the percentage denaturation. When there is a mobile equilibrium between the native and denatured forms of a protein, denaturation is caused by the addition of any substance which has a greater affinity for the denatured than for the native form. Theoretically the heat of denaturation must vary with the denaturing agent and must depend on the heat of combination of the denaturing agent with the protein.     

THE CHANGE IN STATE OF THE PROTEINS OF MUSCLE IN RIGOR

1. When myosin is exposed to a typical denaturing agent (acid) it becomes insoluble and its SH groups are activated. 2. The same number of active SH groups is found in the soluble myosin of resting muscle as in the insoluble myosin of muscle in rigor. No activation of SH groups accompanies the formation of insoluble protein in rigor. 3. When the insoluble myosin of muscle in rigor is treated with a denaturing agent its SH groups are activated. 4. Protein coagulation as brought about by denaturing agents (heat, acid, alkali, alcohol, urea, salicylate, surface forces, ultraviolet light) is a distinctly different change from the coagulation of myosin brought about by the unknown agent in muscle.     

THE REVERSAL OF MITOCHONDRIAL MEMBRANE

An electron microscope study of mitochondria in hamster liver and kidney cells has revealed that at some points the outer membrane of these organelles is continuous with the inner membrane. Also, at such points the discontinuous components of the membrane pairs have free endings. The outer and the inner membranes of a mitochondrion, therefore, may not be two different and distinct entities, as has been conventionally assumed, but may rather be a part of the same unit. Such a morphological structure would make the intramitochondrial substance accessible to the cytoplasmic substance through the intermembrane channel. This structure would also facilitate the swelling of a mitochondrion either by an unfolding of the cristae, or a sliding of the two membranes, or by both these processes occurring simultaneously.     

STUDIES ON BLOOD COAGULATION : II. THE FORMATION OF FIBRIN FROM THROMBIN AND FIBRINOGEN

Although calcium is essential for the formation of thrombin, it can be recovered quantitatively from formed horse thrombin without affecting its coagulating activity. Citrate also has no significant effect. As stated in the text, this does not exclude the possibility that thrombin is actually a calcium compound present in minute concentration; but confirming the results of Hammarsten, it does show that fibrin cannot be a calcium-protein compound unless one assumes molecular weights for fibrinogen greater than 1,000,000. Although the available experimental data concerning the properties of thrombin, the kinetics of its reaction with fibrinogen, and the quantitative relationships between the two do not allow a definite decision as to whether thrombin is an enzyme analogous to rennin, or whether it combines with fibrinogen to form an insoluble compound, fibrin, the weight of evidence does favor the enzyme theory. A given quantity of thrombin can form at least 200 times its weight of fibrin, and in view of the crudeness of the preparation this ratio is probably many times greater. There is no apparent stoichiometric relationship between thrombin and fibrinogen, and thrombin does not disappear from a mixture of the two until the moment of coagulation; the quantity which then disappears is many times the minimal quantity necessary to form the amount of fibrin produced.