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A reproducible technic is presented for the staining of lipoproteins on paper. This method gives a greater total uptake of dye than previously described procedures, especially by the α-lipoproteins. The staining is accomplished with a solution of Sudan Black B in a mixture of methyl and isopropyl alcohols and the excess dye is removed by wash solution of 30% isopropanol.

Investigation of various steps involved in separation of serum proteins by paper electrophoresis and their quantitation by dyeing with bromphenol blue has revealed that the results obtained are quite dependent on a number of variables. It is essential, therefore, that rigid control of these variables be exercised and that normal values be obtained by the specific technic adopted. Normal values have been given for the technic employed and data on stability of sera at room and refrigerator temperatures have been presented.

1. A procedure for paper electrophoresis has been described which gives highly reproducible protein patterns with good resolution and freedom from distortions. 2. Densitometry of protein bands on paper stained with bromphenol blue or Amidoschwarz 10B reveals that the logarithm of protein concentration is proportional to optical density and that Beer's law does not apply. Electrophoretic patterns of normal human serum evaluated in this manner give values in close agreement with those obtained by moving-boundary electrophoresis. 3. Confidence limits were determined for both methods.

1. A yellow-brown component of commercial oil red O separated by our technic of paper chromatography is shown to stain human serum proteins, particularly those that are coagulated by heat. The patterns of serum electropherograms obtained by coloring with the crude oil red O solution consist of a red lipid pattern superimposed on a brownish protein pattern. 2. A method is described for partial removal of the nonred components from the crude oil red O. Satisfactory coloration of lipoproteins was obtained with the "purified" oil red O. 3. It is demonstrated that areas of paper strip covered with protein may stain lighter with some oil red O fractions than the background filter paper.

With use of the serum protein pattern as a model, we compared the "Diagnostic Effectiveness" of tests of different chemical specificity—i.e., the percentage classified correctly according to the clinical diagnosis. When results obtained from a selected population of subjects with selected diseases were evaluated by multivariate analysis, disease discrimination by paper electrophoresis, which resolves only chemically heterogeneous fractions, was similar to that of a battery of specific assays for individual proteins.

Instrumentation is described for the computation of stained paper electrophoresis patterns of serum proteins. The unit not only plots the dyed protein fractions, but with its electronic units, stores the individual integration voltages, scales the voltages to a percentage of the total, and plots the data on a bar graph. By including the total protein value obtained by chemical analysis, a second survey of the integrator voltages provides the concentrations of the protein fractions in grams per 100 ml. of serum.

Analyses by paper electrophoresis showed three protein fractions present in all stages of development except the egg, where only a single fraction was recovered. The fraction with the second greatest mobility was the most variable during the insect's development, always reaching a peak of concentration at and around times of molting, and disappearing between molts; it disappeared in adult males after 3 to 4 weeks and in adult females after 10 days. The first and third fractions showed variability that could be associated with development, but less strikingly. Fraction 3 also was present in greater proportion in adult females than in adult males.

Chlorine and sulfur are assayed in serum with the X-ray spectrometer. The serum samples are dried in confined spots on a long strip of filter paper. The strip is passed continuously through the X-ray field and the element assayed by the peak of the curve drawn on the recorder. An attachment for the spectrometer is described for this purpose, as well as an instrument for making the confined spots. Results for chlorine in serum are compared with those obtained by mercurimetric titration. The results are comparable as regards accuracy and reproducibility. Using 20 µ1. of serum and the automatic feed, the results for chlorine indicate a reproducibility, by X-ray, of ± 3.4 per cent (2σ). No significant difference was observed in comparing the values for sulfur obtained by X-ray with those obtained by ashing and treatment with barium chloranilate. The X-ray technic with 25 µ1. of serum is reproducible to ± 2.2 per cent (2σ). Serum was dialyzed against saline and assayed for sulfur content before and after dialysis. The dialyzable sulfur is small in comparison with the total protein sulfur. In comparing normal and pathologic sera with respect to their sulfur-to-protein content ratio it is observed that this varies apparently because the serum globulins contain amounts of sulfur different from serum albumin. The abnormal protein observed in multiple myeloma contains less than half the sulfur of the albumin from the same patient. This was determined by scanning the electrophoretic pattern of the serum for sulfur and protein. It is pointed out that the X-ray technic is practical for routine analysis in the clinical laboratory in that no reagents are required for total sulfur, chlorine, calcium and potassium; furthermore, only an ultramicrosample is needed and the instrument is easily adapted to automatic operation. Results are comparable with those obtained by conventional methods with regards to accuracy and precision.

Human serum protein fractions were separated by continuous-flow electrophoresis and purified by dialysis, lipid extraction, lyophilization, and drying to constant weight. Increasing amounts of each of the protein fractions were applied to strips of filter paper. One set of strips was stained with amido black 10B dye, and another set with bromphenol blue. The dye-binding properties of the protein fractions were evaluated by (1) the Grassmann-Hannig and (2) Spinco tech nics of direct photometry, and (3) by elution procedures. The calibration charts indicated that dye-binding is not a strictly linear function of protein concentration. This deviation is especially marked in the range from 0 to 40 µg. of protein. Deviation from the Beer-Lambert law with small quantities of protein leads to artifactitious augmentation in the estimation of proteins present in serum in low concentration, such as the alpha globulins. In the range from 40 to 80 µg. of protein, with the three methods for quantitation, the affinities of alpha, beta, and gamma globulin for amido black averaged 80, 71, and 82 per cent, respectively, of that of albumin. Under comparable conditions, the affinities of alpha, beta, and gamma globulins for bromphenol blue averaged 64, 58, and 60 per cent, respectively. On the basis of these findings, amido black 10B appears to have a theoretical superiority over bromphenol blue for use in quantitative estimations of serum protein fractions by paper electrophoresis.

Preparation of Muscle Hemoglobin.-The method of preparing muscle hemoglobin may be described as follows: The animal is freed from blood by the technic used in other experiments to wash out plasma proteins. Under ether anesthesia cannulas are tied into the carotid and jugular vessels. A Ringer's solution with 5% glucose at body temperature is injected by gravity into the jugular vein at the same speed as blood is drawn from the carotid. By slow and careful simultaneous bleeding and transfusion one can reduce the red cell hematocrit from 50% (or normal) to very low values of '2 to 3%. When the heart stops the thorax is opened immediately, a cannula placed in the first part of the aorta, the right auricle incised and gravity perfusion continued freely at a pressure of about 100 cm. of solution for a few minutes, utilizing 3 to 4 liters of perfusate. The two instances in which the muscle hemoglobin solutions gave slight precipitin reactions for dog blood hemoglobin were experiments in which this gravity perfusion was omitted. The procedure obviously washes out the last traces of circulating blood. Massage of trunk, back, thorax and extremities is continued during the last perfusion period. Then the thoracic and abdominal viscera are rapidly removed and further massage of the muscles is continued to remove any traces of perfusate. The various muscles are then dissected free with no loss of time, rinsed and massaged in physiologic salt solution. The tissue is then dried with a towel and care is taken to remove any gross fat or fascia. The bloodfree muscles are ground in a meat grinder and the tissue diluted with four volumes by weight of distilled water or 0.4% ammonia solution. The ammonia solution gives more complete extraction of the hemoglobin from the muscle. The mixture is thoroughly stirred and placed in an icebox (2 to 4 C.) over night. This red solution is now strained through cloth and cleared in large part by centrifugalization which also remove troublesome fat, then filtered through paper. The solution may then be passed through a coarse Berkefeld filter and finally through a finer one to remove any bacteria remaining. This last procedure removes a good deal of muscle hemoglobin but is necessary for certain experiments. The solutions of muscle hemoglobin used in these experiments contained from 600 to 800 mgm. muscle hemoglobin per 100 gmin. muscle. The sterile clear red solution was then shipped in sealed containers by mail for the precipitin tests to be described. A general description of the various methods used for analysis is given in a recent paper.'

Immunoelectrophoretic identification of components of serum protein has become a common laboratory procedure. The technic with agar gel is relatively cumbersome. Modification to permit its performance on a paper-like medium such as cellulose-acetate offers advantages of convenience and uniformity. Cellulose-acetate membrane has been described for electrophoretic separation of proteins ' ' 10 and for Immunoelectrophoresis.-' 6 '-''Sudhof and Walter detailed patterns of 5 serum fractions by means of Immunoelectrophoresis on this medium; however, more detailed immunoelectrophoretic analyses of human serum proteins on cellulose-acetate have not been reported. Inasmuch as immunoelectrophoretic patterns in agar gel and on celluloseacetate would not necessarily be the same, the study described in this paper was made in order to determine the electrophoretic patterns of human serums on that medium.

THE SPECIES specificity of blood protein fractions and the use of these patterns as indicators of relationship between and among different vertebrate taxa has been reported or suggested by several authors (Deutsch and Goodloe, 1945; Dessauer and Fox, 1956; Johnson and Wicks, 1959). These papers have dealt primarily with investigations of the value of protein patterns by paper electrophoresis in separating various taxa. More recently Zweig and Crenshaw (1957) and Dessauer and Fox (1956, 1958) have shown that geographical races of turtles, snakes, and some amphibians can be differentiated by quantitative and qualitative differences in their plasma protein patterns. The potential value of this technique as an objective method of differentiating between and among species has been mentioned by several of the above authors. While the objectivity is not absolute in that further extrapolation of the data is necessary, it precludes the somewhat arbitrary selection of "significant" morphological characteristics on which relationship is based. The studies that have been conducted on the blood protein fractions of insects are for the most part restricted to a few individuals representing distantly related taxa. Electrophoretic separation of insect blood protein fractions has indicated the potential value of this tool in entomological systematics (Clark and Ball, 1957; Stephen and Steinhauer, 1957; Stephen, 1958; Brezner and Enns, 1959). The small

Investigations on the biochemical composition of genital tract secretions during oestrus and early reproductive stages reveal a spectrum of different substrate and time specific patterns, particularly of secretory proteins. Results are presented on the biochemistry, immunological specificity, and endocrinological regulation of rabbit female genital tract secretion proteins. Special attention is drawn upon uteroglobin, which is one of the genital tract specific proteins presumably involved in preimplantational blastocyst development. Since analytical data on rabbit genital tract secretion proteins accumulate, there is consequently a necessity for an approach to comparable studies in the human uterus. Discelectrophoretical patterns and immunochemical test results are presented in this paper which encourage further studies on human genital tract specific proteins with regard to human reproduction, particularly with its contraceptive and fertility problems.

1. 1) Quantitative fractionation of the serum proteins was carried out in brucellosis by the filter paper electrophoretic method. The changes in the serum protein fractions were followed in patients suffering from Brucella melitensis infection, either in its acute stages or during relapses. These changes were also followed in guinea-pigs experimentally infected with Br. abortus injected intramuscularly, and the results compared with normal control healthy animals kept under identical conditions. In both species, the presence or absence of infection with this organism was arrived at by blood cultures and/or serum agglutination tests. 2. 2) It was found that, after infection with Brucella , there was little change in the level of the total serum proteins with the progress of the disease. The albumin fraction showed some progressive decrease as the disease became chronic. The α-globulin fraction rose during the acute stage of the disease, but then came back to normal as it went into the chronic stage; the β-globulin fraction, however, showed a change in the opposite direction. There was a significant progressive rise in the γ-globulin fraction with the advance of the infection, whereas the albumin-globulin ratio showed a progressive fall. The significance of these findings is discussed.

relationships has led to the study of many characters. In addition to comparative morphology some systematists have utilized ecology, behavior, serology, and biochemistry as clues to the degrees of genetic relatedness among organisms. Because protein molecules are primary gene products it is logical to assume that comparisons among homologous proteins from different organisms should provide useful systematic data. The rationale behind this approach to systematics has been discussed by several authors including Sibley (1960, 1962, 1964, 1965, 1967), Zuckerkandl and Pauling (1965), and Dessauer (1969). The plasma proteins are an obvious choice for investigation because they are easy to collect and because a great deal is known about their properties and functions. Plasma is the fluid portion of blood in which the blood cells are suspended. It is a complex mixture of proteins, carbohydrates, lipids, steroids, and free ions whose composition varies with sex, age, starvation, season, etc. (Moore 1948; Clegg et al. 1951; Vanstone et al. 1955; Dessauer and Fox 1956; Saito 1957b). The protein constituents of plasma, while often quantitatively variable, usually show a high degree of qualitative species specificity when examined by any standard biochemical technique (Morris and Courtice 1955; Zweig and Crenshaw 1957; Drilhon et al. 1958; Woods et al. 1958; Sulya et al. 1961). Some of these studies were based upon serum, the fluid portion of the plasma which is extruded from a blood clot. Plasma thus contains the blood proteins involved in clotting while serum lacks them. Most of the research on plasma proteins has dealt with human material although there has been a great deal of work on other mammalian species and the domestic fowl, Gallus gallus. The major protein components of plasma are albumin, the alpha-, beta-, and gamma-globulins, and various subfractions thereof. The nomenclature of the various components is usually determined by their electrophoretic mobilities with reference to normal human plasma. Thus, albumin is the fastest fraction, alpha-globulin the next fastest and gammaglobulin the slowest. The identification of plasma proteins under different conditions can be difficult (Espinosa 1961; Beaton et al. 1961). Up to 70 different proteins have been found in normal human plasma (Dessauer and Fox 1964) while Baker et al. (1966) found 40 electrophoretic bands in pheasant serum, 14 of which were identifiable. The review by Putnam (1960) provides information on the chemical composition of plasma. In paper electrophoresis at pH 8.6 the fastest c mponent in human plasma is albumin. It has a molecular weight of about 69,000 and is is electric at pH 4.7 (Phelps and Putnam 1960). It may be assumed that the values for other mammals and for birds are similar (Phelps and Putnam 1960:169). Plasma albumin has the same chemical properties as alpha livetin of egg yolk (Williams 1962a) but it has no known specific biological functions (Foster 1960). There are several alpha-globulins, which presumably have different functions. The best known are the hemoglobin-binding haptoglobins and the copper-binding ceruloplasmin. Human haptoglobin has a molecular weight of 85,000 and it is isoelectric at pH 4.1, while ceruloplasmin has a molecular weight of 151,000 and is isoelectric at pH 4.4 (Phelps and Putnam 1960). There are several discrete beta-globulins, most of which have unknown functions. The best known of these are the transferrins. Tra sferrin, also called siderophilin, is an ironbinding protein with a molecular weight of approximately 90,000; it is isoelectric at pH 5.9 (Phelps and Putnam 1960). It has been found that the protein moiety of the transferrin molecule is identical to that of the conalbumin of egg white. They differ only in their 1 Present address: Department of Biology, University of North Carolina at Greensboro, Greensboro, North Carolina 27412.

In previous papers in this series1-3 a method has been described for the in vivo production of a protective antigen against infection with Bacillus anthracis. This preparation, however, is not ideal as an immunizing agent when used to immunize other than homologous species because it contains large amounts of sensitizing proteins. Recently Gladstone4 has prepared a protective antigen in vitro which has similar properties to the antigen isolated from crude extracts of edematous lesions.2 He employed whole serum as the substrate, and as a result, like the in vivo preparation, it contains large quantities of serum proteins. His method has an advantage over the in vivo technique in that it is possible to use homologous serum, thus avoiding untoward reactions. Of equal importance, this finding makes possible the eventual isolation and characterization of the factors essential for antigen production by the organism. In this event, it should be possible to produce the antigen in a medium free of serum

Using the combined technics of the ionographic separation of serum proteins in a paper-stabilized medium, as described by McDonald et al. (16, 17, 18), and the spectrophotometric procedure for the determination of transaminase activity developed by Karmen (3, 14), the electrophoretic migration pattern of the enzyme glutamic oxalacetic transaminase in rat serum has been examined. The major portion of the transaminase activity has been found to be associated with the α-2 globulin fraction of the serum proteins. Further evidence has been presented for the assumption of a nonionic linkage between the enzyme and its coenzyme, pyridoxal phosphate.

Seromucoid and serum total glycoproteins were measured in 7 patients with myocardial infarction. In all cases the highest concentrations of these proteins were reached several days after the concentration of glutamic-oxalacetic transaminase had begun to return to normal. In 2 cases paper electrophoretic analysis of the serum proteins was performed. A rise in the concentration of 1- and 2-globulins occurred after infarction, and PAS staining indicated that the rise in total glycoprotein was largely or entirely accounted for by these two fractions. The concentration of serum albumin fell considerably after infarction, reaching a minimum at about 6 days; - and -globulin concentrations appeared to fall slightly. In general, the presence of C-reactive protein corresponded roughly with the period during which the seromucoid concentration was elevated. It is suggested that the elevation of seromucoid and total glycoproteins represents a nonspecific response to the tissue injury.

A modification of the starch-gel electrophoresis technic of Smithies is presented,which enables quantitation of the resolved fractions to be carried out with little equipment other than that used for paper electrophoretic fractionation of proteins.A sheet of plastic (Stabilene film) wrapped around a kymograph type drum is evenly coated with starch-gel. The sheet is suspended in an "open strip" Durrum type cell and electrophoresis carried out. After staining with suitable stain to demonstrate proteins, lipids, or other components, the patterns are scanned in a photoelectric densitometer. Results obtained with this procedure show good reproducibility when fractionating solutions of hemoglobin and serum proteins.

Halogenated acetates alone and in combination with dextrose and formaldehyde will protect against heat denaturation of plasma proteins even when plasma is heated for 10 hr. at 60°. Application of this principle would afford reclamation of plasma from outdated blood bank blood. Changes in protein patterns were observed by paper electrophoresis. Other measurements of denaturation were pH, absorbance at 610 mµ, and viscosity. The chemical reaction between the additives and plasma are considered, and estimations of toxicity are given.

The paper describes a method for the estimation of azovan (Evans) blue in plasma proteins precipitated by trichloroacetic acid. The precipitate is dissolved in a strong solution of urea and an anionic detergent; the dye is absorbed on a cellulose column, from which it can be eluted by alkaline acetone-water. The method allows information to be obtained about drug uptake and plasma volume without the need for additional blood samples. It has been used with cat, rabbit, rat and guinea-pig plasma.

of laked blood. In these experiments agglutinins and lysins for red blood cells have been considered as well as precipitins. Rabbits 1, 2 and 3 were injected with a mixture of equal amounts of beef, chicken, dog, guinea pig, horse, human, monkey, rabbit, rat and sheep blood; in rabbits 4 to 8, hog blood was substituted for monkey blood. The mixture was prepared as follows: some 30 to 40 cc. of each blood was added to 5 cc. of a 1 per cent sodium citrate solution and shaken well to prevent clotting; approximately 10 cc. of each of the ten samples of blood were added to 90 cc. of sterile distilled water and placed in the icebox over night. To make the mixture isotonic 0.9 gm. of sodium chloride was added to each 100 cc. of laked blood. The ten samples were pooled and centrifuged to remove the stroma of the red blood cells. The stroma was placed in a sterile mortar and ground with sterile sand. The sand was washed to remove all material possible and this was added to the pooled blood mixture. The entire mixture was put through filter paper to remove all large particles. To prevent the growth of bacteria, 0.3 per cent chloroform was added. The rabbits were injected intravenously with 2, 4 and 6 cc. and then intramuscularly with the 8 and 10 cc. of the mixture. The injections were given at intervals of three to four days. All the rabbits developed precipitins for the mixture and precipitins were demonstrated to be present in their serum for almost all of the blood proteins and hemoglobins included in the mixture as well as for the serums in question (table 1). No precipitins were found for rabbit hemoglobin or serum. It was found also that the eight rabbits had developed agglutinins and lysins for the red corpuscles in varying degree in many instances and most marked in the case of chicken, horse and sheep blood (table 2). Absorption experiments were made with some of these complex antiserums. Serums 1, 2 and 3 were absorbed with guinea pig hemoglobin. One