In this paper a structural theory of protein denaturation and coagulation is presented. Since denaturation is a fundamental property of a large group of proteins, a theory of denaturation is essentially a general theory of the structure of native and denatured proteins. In its present form our theory is definite and detailed in some respects and vague in others; refinement in regard to the latter could be achieved on the basis of the results of experiments which the theory suggests. The theory (some features of which have been proposed by other investigators) provides a simple structural interpretation not only of the phenomena connected with denaturation and coagulation which are usually discussed (specificity, solubility, etc.) but also of others, such as the availability of groups, the entropy of denaturation, the effect of ultra-violet light, the heat of activation and its dependence on pH, coagulation through dehydration, etc.I. The experimental basis upon which the present theory rests will be briefly described.1. The most significant change that occurs in denaturation is the loss of certain highly specific properties by the native protein. Specific differences between members of a series of related native proteins and specific enzymatic activities of native proteins disappear on denaturation, as the following observations demonstrate:(a) Many native proteins can be crystallized and the crystal form is characteristic of each protein. No denatured protein has been crystallized.
A number of methods have been described for the estimation of peptic activity, many of which are accurate and convenient for comparative experiments but none of which give results which are accurately reproducible with different preparations of the protein used. This variation is due to the difficulty involved in obtaining reproducible protein preparations and keeping them unchanged. Hemoglobin has been chosen as the protein substrata for the estimation of pepsin because it is easily prepared in large quantities, because it can be stored in solution for a long time without change, because it is rapidly digested, and because the rate at which it is digested by a given pepsin solution does not vary from one hemoglobin preparation to another. Not only is hemoglobin a reproducible protein which can be brought to reproducible conditions but the conditions of digestion have been so chosen that reasonable variations in these conditions have little effect on the amount of digestion. In general the procedure used to estimate pepsin is this. A pepsin solution is added to acidified hemoglobin. After 5 minutes trichloracetic acid is added. The resulting precipitate which contains all the pigment and all the undigested hemoglobin is faltered off. The filtrate contains an amount of digested hemoglobin which is a measure of the amount of pepsin used. This digested hemoglobin is estimated by the blue color it gives with the phenol reagent, which reacts with tyrosine, tryptophane and cysteine groups, tyrosine being used as a standard. The intensity of this color is proportional to the amount of enzyme used and to the time of digestion.
The DNA (desoxyribonucleic acid) content per cell, according to some recent investigations, is a constant for the various somatic cells of an organism, and sperm cells contain one-hail this amount per ceU (1, 2). The quantity of DNA per cell is a characteristic of each organism. In this work, done independentiy by two groups of investigators, the DNA per ceLl was found by determining the quantity of DNA in a suspension containing a known number of ceUs and then dividing the total DNA by the number of cells. For sperm and erythrocytes the cells themselves were counted; for tissue cells isolated nuclei were prepared, analyzed, and counted.Knowledge concerning the DNA content per cell has been extended along two lines. On the one hand, more examples have been brought forward. In the original work, for example, the DNA contents of erythrocytes, liver ceUs, and sperm of fowl were determined and DNA determinations for cells of fowl kidney, spleen, heart, and pancreas have now been added by Davidson and his colleagues (3). These results are given in Table I. In a series of careful measurements by Davison and Osgood on human granulocytes and lymphocytes (from leukemic blood), two quite different cell types, the DNA per cell of the former was found to be 6.25 X 10 -0 rag. and that of the latter 5.84 × 10 -0 rag. (4). Our own determinations on human sperm gave 2.72 X 10 -9 mg. per cell, approximately one-half the value for the somatic cells.Another line of investigation has been to determine by a cytochemical procedure the DNA content per nucleus (5). In this procedure it is possible to make determinations on single nuclei, either as isolated cells and nuclei or in tissue sections. It has been found that the Feulgen nucleal reaction gives reproducible results and that a microphotometric observation serves as a measure of DNA content. The essential requirement in this procedure is a standard of known DNA content and heretofore none was available. A whole series of standards was provided by the work mentioned above in which DNA per nucleus was determined chemically. One set of standards consisted of nucleated * Dr. Ris' present address is:
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