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The electrophoretic behavior of serum leucine aminopeptidase (LAP) in normal individuals, in pregnancy, and in patients with hepatobiliary disease was investigated with paper electrophoresis technics. In normal serum LAP migrated as a single component in the α1-globulin fraction. In patients with elevated serum LAP activity due to hepatobiliary or pancreatic disease supplementary peaks of enzymatic activity were demonstrable in other protein fractions. Thus, in jaundiced patients with carcinoma of the head of the pancreas, liver metastases, biliary atresia, acute hepatitis, and drug-induced jaundice, additional activity was observed in the β-globulin fraction. In some patients with hepatitis additional activity was observed in the α2-globulin as well as β-globulin. In common duct stone additional activity in the albumin fraction was a consistent finding. In early pregnancy the electrophoretic pattern of LAP was similar to that observed in normal serum, whereas during the third trimester LAP activity was demonstrable only in the α2-globulin.The valuable technical assistance of Mrs. Sally Spurling and Mrs. Thelma Alpren is acknowledged.

Simultaneous use of paper electrophoresis and radioactive Vit. B12permits separation and quantitation of the various B12binding components present in commercial intrinsic factor concentrates of hog origin. The amount of one particular binder has been found to correlate with the clinical activity of the concentrates as measured by the urinary excretion method of Schilling. This provides the basis for a simple in vitroassay of potency of hog intrinsic factor concentrates ranging in level of activity from 2.0 mg to 75.0 mg. It is predicted that pure hog intrinsic factor will be active clinically at a level of 0.1 mg; calculations suggest that it will bind 23 μg B12/mg and have a molecular weight of 60,000.

1. An albumin and 5 globulin fractions have been observed on electropherograms of sera of sexually immature pullets. The albumin fraction of such sera was associated with lipid, but lipid was not observed in association with the albumin of sera of estrogenized pullets. 2. Zones of the electropherograms of sera of pullets that stained with Oil Red O also gave the Feulgen plasmalogen reaction. The Feulgen reaction was stronger with sera of estrogenized birds. 3. Electropherograms of yolk dispersed in saline were prepared using 20% methanolic veronal buffer. A presumptive phosphoprotein fraction was distinguishable on these papers; it resembled closely the presumptive phosphoprotein fraction of sera of laying hens or estrogenized immature pullets. The presumptive phosphoprotein zones were doubled for both yolk and sera. 4. Studies of electropherograms of estrogenized immature pullets that had received injections of phosphate labeled with P32showed that a large moiety of the phospholipid moved ahead of the phosphoprotein fraction when methanolic buffer had been used to separate this PP fraction, although a part of the phospholipid did remain in association with the PP fraction.

Using paper electrophoresis, satisfactory separation of protein components in salivary secretions of the rat was most conveniently obtained with parotid saliva. This secretion was readily obtained in uncontaminated form from the cut surface of the exteriorized parotid duct and required neither concentration nor dialysis for satisfactory electrophoresis. Quantitative variation in total protein and electrophoretic patterns was small between secretions of the right and left glands of rats of an inbred strain and a randomly-bred strain. The variation between rats of an inbred strain, in contrast to a randomly-bred strain, was generally of the same order of magnitude as variation between right and left glands.

Differences have been found both in the number of components electrophoretically separable from individual animal hemoglobins and in the mobility of these components relative to human A, S, and C hemoglobins. Under the conditions employed, with the exception of the horse, 2 of 3 calves, and 2 of 6 sheep, all of which possessed 2 components, the (adult) mammalian hemoglobins studied were found to have single components. Some displayed anodic mobility similar to that of human A hemoglobin (chimpanzee, rhesus monkey, pig, sheep, goat, cat, mouse, rat, hamster), others had greater mobility (macaque, guinea pig), and some possessed lesser mobility (llama, cow, dog, rabbit). In the Hereford steer, it would appear that temporary persistence of fetal hemoglobin accounts for the finding of 2-component hemoglobin in the calf and single-component hemoglobin in adults. Hemoglobin from several New Hampshire chicken and one Leghorn was found to have 3 electrophoretic components; that from other Leghorns, ducks, and a robin showed 2 components. Penguin and pigeon hemoglobin had single components. Hemoglobin prepared from each of 3 species of turtles possessed 2 widely separated bands. Black snake, water snake, and toad had one-component hemoglobins. Frog hemoglobin gave a 2-component pattern. With few exceptions the number of individual hemoglobins tested in any species has to date been small. It is likely that the study of larger numbers of animals will uncover additional examples of heritable variations in hemoglobin types.

Pepsin-treated ACTH materials at approximately 10 times standard were fractionated in a chromatopile using the system butanol-acetic acid-water. The activity was found to be associated with a slow-moving ninhydrin-positive area from which fractions having potencies of about 50 times standard were recovered. No further chromatographic resolution of these fractions was obtained in the solvent systems ordinarily used for amino acids and peptides. In all cases, the activity was associated with ninhydrin-positive areas of the chromatograms.

The alpha globulin portions of the electrophoretic patterns of dog sera and plasma have been shown to be affected by the technics of preparation. While “standard serum” patterns show a predominance of alpha-4 over alpha-3 protein and polysaccharide, “sedimented serum” and oxalated or heparinized plasma show greater proportions of alpha-3 components.

1. Phagocytosis of formalin-killed E. coliwas promoted by previous contact with filter paper or with para-hydroxybenzoic acid. The latter, an active principle in filter paper, was effective in concentrations of 1 to 0.001 mg per ml. Fixed tissue inhibited phagocytosis. 2. The activity of the several surfaces reported by Wood et al.to promote phagocytosis may be ascribed to the wet filter paper in their experiments. These observations open to question the phenomenon of “surface” phagocytosis.

Paper electrophoretic studies showed that the rate of migration of beta-lipoproteins was considerably less at 5°C than at summer room temperature or at 35°C. No effect of temperature was evident in the case of the lipoproteins migrating with the albumin, alpha1-globulin. The rate of movement of the main serum proteins was apparently the same at the different temperatures, but the resolution of the alpha2- and beta-globulins was sometimes less distinct at the higher temperature. The results presented show the importance of using relatively constant temperatures when comparative paper electrophoretic studies are carried out, especially of beta-lipoproteins.

At the present time biological assay methods are the sole means of determining penicillin potency. A number of such methods are in use in various laboratories;1however, the Oxford cup method,2with several modifications,3has continued to be regarded as a procedure in which a commensurate accuracy and speed may be obtained with a minimum of labor. The following filter paper disc modification should reduce the labor and time involved in setting up this test, while the case with which extra replicates may be run should increase the test's accuracy.The principal modification lies in the use of a thick filter paper disc saturated with the penicillin sample, substituted for the sample-containing small cylinder used in the Oxford cup method. These discs may be conveniently set up on the seeded plates at a rate of about 6 per minute, a rate considerably more rapid than that in the original method. Another advantage of the discs is that the test plates may be manipulated freely to facilitate reading. Experience with a large number of tests has indicated that the zones of inhibition obtained with the discs are more consistent and more sensitive to variations in the penicillin content of the sample than the zones obtained with the cup method. This improvement may be due to a more consistent contact of the penicillin solution with the agar and to a more even diffusion from the disc.The test organism, Staphylococcus aureusH†(Oxford strain), tends to produce a somewhat granular growth in nutrient broth. With the use of peptone broth as the seeding medium, a more diffuse growth is obtained and this in turn effects a more even seeding of the test plates.

The reaction of Ehrlich's reagent, p-dimethylaminobenzaldehyde, with aromatic amines in acid solution has been made the basis of a colorimetric determination of sulfonamides by Werner1. and by Morris.2Fuller3has employed this substance in test papers for estimating the level of sulfonamide in laked blood after precipitation of proteins with an acid reagent, comparing the yellow color produced with standard color strips. The present paper describes the preparation of acid-containing test papers with which the level of sulfonamide can be estimated directly in serum without the addition of precipitating or acidifying reagents.One gram of p-dimethylaminobenzaldehyde (straw-colored material is usable) is dissolved in 2 cc of concentrated hydrochloric acid and to the solution is added 0.8 cc of syrupy phosphoric acid (sp. gr. about 1.7) and water to make 100 cc. Large pieces of a good grade of absorbent paper (such as is used in making litmus paper) are soaked in the solution and immediately hung up to drain. When superficially dry, the papers are stored in the dark in covered glass jars for from 5 to 10 days, during which time most of the hydrochloric acid evaporates. The edges of the paper, which are usually yellow, are then trimmed off and the paper is cut into small strips and preserved in suitable vials. The strips are almost colorless, but become discolored if they come in contact with the skin at any time during preparation or use. The phosphoric acid remaining in the papers is sufficient to give a pH of approximately 3 when they are moistened with serum.Estimation of the sulfonamide level is made by spreading evenly the serum to be tested over one end of an impregnated strip by means of a fine dropper, care being taken that unabsorbed fluid does not remain.

A procedure has been developed whereby paper chromatograms of antiviral agents can be developed by applying them to virus infected, agar overlay, chick embryo cell tissue cultures. Plaque-free zones are produced, thus indicating the area on paper chromatogram where the active material can be located. Using antiviral substances antibiotic W-112 and isatin-monothiosemicarbazone, W. Nile and vaccinia viruses, and 4 chromato-graphic solvent systems, it was possible to localize the biological activity of these antiviral agents. As little as 1 μg of antibiotic W-112 produced large plaque-free zones when paper chromatogram was applied to the overlay culture 1 hr after virus infection. When 100 μg of same material was used, large plaque-free areas resulted even when the paper was applied 46 hr after virus infection. Isatin-monothiosemicarbazone at 50 μg level not only produced a plaque-free zone but also produced a yellow spot on paper chromatogram that coincided exactly with the zone of antiviral activity.

A procedure for the application of filter paper partition chromatography to the separation of both volatile and non-volatile organic acids with chain lengths of about eight carbons or less is described. The potassium hydroxamate derivatives of the organic acids are prepared by reacting the methyl ester with about a two-fold excess of a mixture of potassium hydroxide and hydroxylamine in methyl alcohol. The hydroxamate derivatives obtained from about 10−6mole of each of the esters are applied to the filter paper, the chromatogram developed with suitable solvents, and then sprayed with ferric chloride to make the derivatives visible as purple spots on a yellow background. Isobutyric acid and phenol gave the best results of the solvents tried for two-dimensional chromatograms of the dicarboxylic acids. RFvalues for a number of hydroxamate derivatives are given for several solvents.

Acid and alkali behave oppositely as far as their extracting power towards an acid and an alkaline dye is concerned. Addition of NaCl inhibits the extracting power of stronger concentrations of acid and in certain cases also that of alkali towards neutral red. Neutral NaCl solutions on the other hand extract neutral red better than water. How far in these effects the dissociation depressing action of NaCl or other actions of NaCl are concerned is uncertain. These experiments demonstrate that there exists a certain similarity between the action of acid and alkali on amcebocytes on the one hand, and on filter paper on the other hand; but this simlarity is incomplete. It therefore appears probable that the conditions determining the staining of cell granules by neutral red differ in some essential respects from those determining the staining of filter paper.

Methods for identification and separation of several non-keto steroids in 10 μg quantities on paper chromatograms are described.

Duodenal fluids from patients with cystic fibrosis of the pancreas and from control subjects were studied by paper strip electrophoresis. Components with greater electrophoretic mobility than any in normal duodenal fluid occurred consistently in cystic fibrosis duodenal fluids. The most rapidly migrating of these fractions was identical in electrophoretic mobility to the albumin fraction of normal human serum.

Serum lactic dehydrogenase from leukemic and non-leukemic individuals has been separated into a number of components by ammonium sulfate concentration gradient elution and by paper electrophoresis. The effect of glutathione and of other proteins upon the fractionation patterns obtained by these two technics is described.

A procedure for the identification and determination of creatine, creatinine, glycocyamidine and glycocyamine is presented. Following resolution by paper chromatography, the developed chromatograms are heated to 100°C for one hour to dehydrate creatine and glycocyamine in situto creatinine and glycocyamidine respectively. The latter compounds and others of similar structure give an orange color on treatment with the Jaffe reagent (alkaline picrate).The Voges-Proskauer reaction (alkaline diacetyl) for creatine was also investigated and found suitable but less precise.RFvalues for creatine, creatinine, glycocyamine, and glycocyamidine are given for water-saturated solutions of butanol, phenol, and lutidine-collidine (50-50).The chromatographic method possesses the advantage that creatine and creatinine can be determined independently and directly rather than by a differential procedure as in the conventional methods.

After treatment of human synovial fluid with hyaluronidase, the hyaluronate and proteins can be separated by paper electrophoresis. The electrophoretic distribution of the protein fractions of normal synovial fluid differs from that of the protein fractions of both normal serum and osteoarthritic synovial fluid.

The number of free amino acids found in unhydrolyzed urine by paper partition chromatography is less than that indicated by microbiological assay. No discrepancy is apparent between the methods when hydrolyzed urines are analyzed. As the sensitivities of the two methods are comparable, it is suggested that microbiological techniques are not as specific as the chromatographic procedure for the detection of urinary amino acids in the uncombined state.

If proteins are added to homogenates of organs before paper electrophoresis, better separation and increased activity of the LDH isozymes is obtained. This increase was confirmed by the spectrophotometric determination of the formazan produced. A mixture of human albumin and gamma globulin in the normal serum ratio (4:1) gives a good electrophoretic resolution of the five isozymes.

The distribution coefficients of amino acids between the 2 phases of a phenol-water mixture have been determined at various H+concentrations, and at temperatures of 20°, 25°, and 30°C. Rf values calculated from each of these distribution coefficients have been correlated by statistical methods with corresponding Rf values as measured by paper chromatography and a significant relationship has been demonstrated.

Thrombokinase prepared from bovine plasma was further purified by continuous flow paper electrophoresis. Thrombokinase was assayed by its capacity to activate prothrombin in the presence of oxalate, and also by production of thrombin in a system containing prothrombin, cephalin, calcium and bovine “barium carbonate serum.” Curves of these 2 assays were similar and formed a peak ahead of the thrombin peak. TAMe esterase activity appeared in 2 peaks which corresponded respectively to thrombin and thrombokinase peaks.

A radioautographic technic with a sensitivity many times greater than conventional staining methods is described for evaluation of paper electrophoretic studies of soluble antigen-antibody complexes. Electrophoretic mobilities of BSA-anti BSA soluble complexes relative to rabbit serum proteins are reported with a semi-quantitative analysis of percentage concentration for each of the complexes at various values of antigen excess.

By use of paper electrophoresis of crude extracts of heart and liver, 2 fractions with GOT activity were obtained, one of which migrated toward the anode and showed the known characteristics of serum GOT, while the other migrated toward the cathode and exhibited different affinities for both α-ketoglutarate and L-aspartate.

A simple, quantitative method for the microbiological assay of filter paper chromatograms has been described. Some possible applications have also been pointed out.

Rats which have developed parabiosis intoxication show, in addition to the other hematologic aberrations which characterize the condition, changes in the blood proteins. Anemic partners have a low plasma protein level and polycythemic animals an elevated plasma protein titre. The partitions which albumin and the respective globulins contribute toward the total serum proteins average the same in both anemic and polycythemic twins, although as compared to control levels there is a diminished serum albumin and an elevated α2-globulin component in each.

Three pairs of male littermate rabbits were fed a commercial vit. E deficient and a vit. E supplemented diet. When the vit. E deficient member of each pair showed typical vit. E deficient symptoms, the pair of animals was sacrificed and the water soluble muscle proteins extracted from the striated muscle. The electrophoretic pattern of the deficient animals showed a significant change in distribution of water soluble proteins as compared to the control group. This change was an increase in the fast moving peak and a decrease in the slow moving peak.

Cortisone administration to dogs is followed by a selective rise in plasma alpha-2 globulin and fall in beta globulin which is independent of any associated lipid changes; this shift in globulin distribution has not been observed in humans or rabbits receiving cortisone.