The isolation of trypsin from active pancreatic extract (1) and of chymo-trypsinogen and chymo-trypsin (2) 1 from fresh inactive pancreas has been described in previous papers. Crystalline trypsinogen has also been obtained (3) from inactive cattle pancreas and transformed into active trypsin which may then be crystallized much more readily than by the earlier method. During the course of this work a polypeptide, which has a powerful inhibiting effect on trypsin, as well as a compound of this substance with trypsin, was also obtained in crystalline form (4). The present paper contains detailed descriptions of methods of preparing these substances and a brief description of their properties. G E N E R A L P~ROPERTIES TrypsinogenTrypsinogen is obtained as small triangular prisms (Fig. 1). When these crystals are dissolved in neutral solution the trypsinogen is rapidly transformed into active trypsin and it has, therefore, been impossible so far to recrystallize trypsinogen. The original crystallization occurs without activation owing to the presence of the inhibitor and if inhibitor is added to a solution of trypsinogen recrystallization may be carried out without activation. Numerous attempts have been made to recrystallize inhibitor-free trypsinogen under conditions 1 It has recently been found that poorly formed needle-shaped protein crystals, which have about the same activity as the usual chymo-trypsin crystals, may be obtained from the mother liquor of the chymo-trypsin crystallization. The properties of these new crystals are now being investigated.
In 1908 one of us raised the question whether there is a definite temperature coefficient for the duration of life.' This might be expected if the duration of life depended upon the presence of certain substances which were used up during life; or if the duration of life were limited by the cumulative injurious effects of certain products of metabolism. Thus, Metchnikoff2 has mentioned the possibility that the duration of the life of the moth of the silk worm is limited by the retention of certain poisonous substances contained in the urine. The rapidity of consumption of the necessary substance in the case of the first or the velocity of the accumulation or action of injurious substances in the case of the second hypothesis should increase with the temperature according to a certain law.It seems that there exists for each species a pretty definite duration of life in spite of the fact that injuries of various types may shorten the life of the individual. The annual plants, the sequoia of the sierras, the human, the insects, have their characteristic duration of life. On the other hand, it was shown by Leo Loeb that the cancer cell is immortal and he pointed out that this might be the case for all cells. Then the problem arises, what is the cause of the fact that each species has a limited duration of life the magnitude of which is characteristic for the species? If the answer to this question is given by one of the two hypotheses mentioned in the first paragraph of this paper, it may be expected that there should be found a temperature coefficient for the duration of life of the order of magnitude of that of chemical reactions. A search for such a temperature coefficient can only be attempted on a form with a naturally short duration of life. We have selected for this purpose the fruit fly Drosophila.Newly hatched flies were put into large Erlenmeyer flasks kept in thermostats 34°, 31°, 28°, 24°, 14°, and 9°. Each flask contained on the average about 100 flies. The number of dead were counted each day and the surviving flies were put into fresh flasks every two days. Each determination of the duration of life was based upon at least two and often as many as twelve cultures of about 100 flies each. The values for the mean length of life of the flies in the separate cultures, at a given 456
A method is described for determining the diffusion coefficient of solutes by determining the rate of passage of the solute through a thin porous membrane between two solutions of different concentration. The method has been used to determine the diffusion coefficient of carbon monoxide hemoglobin. This was found to be 0.0420 ± 0.0005 cm.2 per day at 5°C. The molecular weight of carbon monoxide hemoglobin calculated by means of Einstein's equation from this quantity is 68,600 ± 1,000.
Kigane (1) and Heidenhain (2) showed that extracts of fresh panereas or freshly secreted pancreatic juice have no proteolytic activity. The preparations become active when mixed with the enterokinase of the small intestine, as found by Schepowalnikow (3) or when the pancreas is allowed to stand in slightly acid solution. The mechanism of this activation has been the subject of controversy for many years (4). Pavlov, Bayliss, Zunz, Wohlgemuth, Vernon, Delezenne, and others found the activation reaction to be catalytic and considered enterokinase to be an enzyme. Hamburger and Hekma, Dastre and Stassano and Waldschmldt-Leitz found the reaction to be stoichiometric and considered that the enterokinase formed an addition compound with the inactive zymogen. Vernon (5) found that activation could be caused by trypsin as well as by enterokinase but this was denied by Bayliss and Starling (6). The contradictory nature of the numerous experimental results indicates that there is more than one proteolytic enoune in pancreatic extracts. Vernon showed (7) that the activity, as determined by the clotting of milk, could be partially separated from the proteolytic activity, as determined by protein hydrolysis, and concluded that there were at least two enzymes. He also showed that one of these was more stable than the other and that activation was caused by the less stable one.The crystalline trypsin previously reported by the writers (8) was obtained from pancreas which had been allowed to activate spontaneously. The present experiments were carried out with fresh inactive pancreatic extracts in order to obtain the inactive form of the enzyme and to study the kinetics of activation.
A method is described for the preparation of a crystalline protein from commercial pepsin preparations which has powerful peptic activity. The composition, optical activity, and proteolytic activity of this protein remain constant through seven successive crystallizations. No evidence for the presence of a mixture or of a solid solution is found in a study of the solubility of the protein in a series of different salt solutions, nor from the diffusion coefficient or from the rate of inactivation. These results indicate that the material is a pure substance or possibly a solid solution of two or more substances having nearly the same solubility in all the various solvents studied. It seems reasonable to conclude from these experiments that the possibility of a mixture must be limited to a mixture of proteins, so that the conclusion seems justified that pepsin itself is a protein.
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