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Using a combination of two technics, paper chromatography in the first dimension and paper microelectrophoresis in the second dimension, it is shown that protein separation may take place during capillary ascent of a mixture of proteins on filter paper and that protein interaction may also occur.

Further details are given concerning the movements of proteins on filter paper using M/10 solutions of sucrose solution and sodium potassium tartrate as developing solvents in the first and second dimension, respectively. It is shown that mixtures of proteins, such as serum albumin and cytochrome c, may be separated from each other on paper, since the former moves in the first dimension, whilst the latter does not and both proteins move in the second dimension. The amount of cytochrome c moving in the second dimension is proportional to the amount applied to the paper. The chromatographic patterns of purified proteins e.g.crystalline human serum albumin, α, β, and γ globulins, and fibrinogen differ from each other in a characteristic manner. The differences are more marked when the proteins are mixed with surface active agents with which they form complexes; and these complexes move at different rates on filter paper. Complexes of surface active agents with albumin move with greater ease along the first dimension than those formed with globulins; the latter tend to remain at the point of origin. Almost all the protein complexes, however, move in the second dimension. The pattern given with a blood plasma is a composite of the patterns of the individual protein fractions, the basal fractions being apparently due to globulin. The patterns of chromatograms of pathological cases (multiple myeloma, liver cirrhosis, cancer of the stomach, lupus erythematosis) differ significantly from the normal, and are often characterized by the presence of heavy basal fractions due probably to increase in the globulin content of the plasma.The chromatograms of dried snake venoms (cobra, Russel viper, Agkistrodon piscivorus) show various distinctive features.

Serum was subjected to electrophoresis on filter paper and the distribution of lipid studied by application to the paper strips of Sudan stains, the Schultz test, and other histochemical procedures. Two principal lipid bands, probably corresponding toβandαlipoproteins, were recognized, the larger being in theβ-globulin region, and the smaller in the albumin zone. In some cases lipid apparently associated with little protein was also found at the point of application of the serum. The administration of heparin to human subjects, or its addition to serum, rapidly produced a change in the distribution of sudanophilic and Schultz-reacting lipid.

A continuous paper electro-phoretic technic was compared with a paper strip electrophoretic technic as a tool for the study of serum protein and glycoprotein fractions. Protein and glycoprotein for the continuous electrophoretic study were quanti-tated by analyses of the fractions by the biuret and tryptophan reactions. The glyco-proteins by the strip technic were quantitated by the periodic acid Schiff reaction and the protein strips by the bromophenol blue staining. In both cases the strips were evaluated with an automatic photoelectric scanner. Good agreement between the two methods for both protein and glycoprotein was noted.

1. A new technique of resolving and identifying the members of the B6 family of growth factors is described. It involves the use of paper partition chromatography coupled with a microbial indicator.2. Using this method it has been demonstrated that pyridoxal undergoes a non-enzymatic transamination reaction even in the cold. Pyridoxal also appears to react reversibly with other amino acids.3. The reaction between cysteine and pyridoxal differs from that with other amino acids in that one can demonstrate the formation of at least one adduct which moves more slowly on the chromatogram than does pyridoxamine. This reaction may produce a new form of vitamin B6 which may also have a transient existence in nature.4. The technique described should prove of value as a means of establishing the purity of the various forms of vitamin BG used, in biological and biochemical studies.5. The general technique employing paper partition chromatography and microbial indicators should be of value in resolving other multiform vitamins into their constituent parts.

The effect of temperature on velocity of electromigration through paper stabilized electrolytes, of a variety of substances, including bromphenol blue, bovine plasma albumin, human serum albumin and globulin fractions and human serum lipoproteins, was investigated. The ionographic apparatus employed utilized horizontal paper strips in a water saturated atmosphere. The runs were made at temperatures from 4°C to 25°C and under constant conditions of buffer, ionic strength, pH and potential gradient. For all substances studied, the relationship between the mobility and temperature can be represented by the equation:where T is ° Kelvin, B is a constant and m is the slope of the line obtained when the logarithm of the mobility is plotted against the reciprocal of the absolute temperature.

1. Results of paper electrophoresis of mixtures of radioactive vit. B12and concentrated human gastric juice demonstrate that the radioactive B12 is bound to a component of gastric juice which is not one of the major protein peaks and moves toward the anode. This behavior of the vit. B12was not noted when it was mixed with saline or serum. 2. This localization of vit. B12to a specific portion of gastric juice by paper electrophoresis may be a clue to the further identification of the intrinsic factor of Castle.

Nine serum protein fractionation experiments using the continuous flow paper electrophoresis technique were performed. Six experiments were performed using the serum collected from individual patients 24 and 48 hours after receiving therapeutic doses of I-131 and 3 using the serum of euthyroid individuals which was incubated in vitrofor 1/2 hour at 37°C with I–131 labelled thyroxine prior to fractionation. Protein fractions obtained were identified by conventional paper electrophoresis and analyzed for radioactivity, total protein and cholesterol, the latter being taken as an index of lipid contents. Similar results were obtained for both in vivoand in vitroexperiments. Beta globulins were found to be capable of forming thyroid hormone protein complexes to a larger extent than gamma globulins, but the largest amount of radioactive hormone was found as an alpha-2 globulin-thyroid hormone complex. A patient showing a marked abnormality in his plasma proteins showed the highest percent of radioactive hormone as protein thyroid hormone complex with the mobility of albumin. The highest percent of the radioactivity was found in fractions having a low protein concentration, indicating that the existence of a protein thyroid hormone complex does not depend on the abundance of a particular protein but on the number of specific binding sites that a particular protein may have and on thermodynamic factors influencing the association constant of the complex so formed. It was also found that the fractions having the highest cholesterol level and hence higher lipid contents contained little radioactivity, indicating that the bulk of thyroid hormone binding is accomplished by a lipid poor protein.

1. The medium in which B. abortusstrain 19 was grown influenced the phagocytosis of this microorganism. Brucellaegrown on tryptose agar were phagocytosed more readily than those grown on beef agar, or in tryptose or beef broth. The effects of the media were different with Brucellaand E. coli.2. Phagocytosis of formalin-killed B. abortus19 was promoted by previous contact with filter paper or para-hydroxybenzoicacid. The latter was effective in concentrations of 1.0 to 0.01 mg per ml, and the stimulatory effect was related in linear fashion to the logarithm of the concentration of para-hydroxybenzoicacid. These effects of filter paper and para- hydroxybenzoic acid with killed microorganisms were similar to those observed with E. coli.

1. Changes in the plasma protein patterns of dogs after high fat meals, and after the intravenous administration of heparin or Tween 81, have been demonstrated, using the paper chromatographic technic of Franklin and Quastel. 2. Concurrent reductions in the hematocrit, and changes in the sedimentation rate were observed after these procedures. The chylomicron counts were decreased after intravenous heparin. 3. The evidence indicates that the surface active substances present in the blood are modified after the above administrations, thus affecting the protein patterns of the paper chromatograms.

The paper electrophoretic pattern of human serum has a general resemblance to other mammalian species in number of components and their electrophoretic properties. Globulin and albumin fractions of avian species (domestic fowl, cormorant) resemble in mobility those of the human. Serum components of lower vertebrates differed in their electrophoretic properties from those of the human and thus are not comparable. The serum pattern from an anthropod, H. americanus, was completely different from the patterns obtained from vertebrates.

By use of 30% Amberlite IRC 50 (W.A. 1) resin-exchange paper, it is possible to separate both insulin and glucagon independently from their mechanical mixtures with non-specific serum or gamma globulin.

Two methods are described which permit the estimation of colored substances on one-dimensional and two-dimensional paper chromatograms with a minimum expenditure of time and material.

1. Paper electrophoresis provides a simple reproducible method for the study of serum (plasma) lipoproteins. Characteristic species lipoprotein patterns were demonstrated in normal man, monkey, dog and rabbit. 2. This method combined with chemical determinations of serum (plasma) lipids permits detailed investigations of normal and abnormal lipid metabolic states. 3. Elevation of serum (plasma) lipid fractions induced in these species by various experimental procedures were accompanied by a lowering of α lipoprotein and varying degrees of elevation of the β + O fraction.

A technic for the two dimensional chromatography of blood plasma and protein mixtures on filter paper is described. Separations of protein constituents are greatly facilitated by the addition of surface active substances such as the “Tweens” or “Spans,” etc. The technic at present adopted is to add “Tween 85” or “Tween 81” and, using hemin as protein marker, to employ M/10 sucrose solution in the first dimension and M/10 sodium potassium tartrate solution in the second dimension. Differences between the protein patterns of chromatograms of human, rat and guinea pig plasma are noted. With protein mixtures, associations of proteins may occur that may be detected by chromatography. The sera of hyperimmunized horses (as against diphtheria toxin) yield chromatograms which differ from, and exhibit fractions not present in, chromatograms of normal horse sera.Grateful acknowledgment is made to the National Cancer Institute of Canada for a grant in aid of this work, to the Department of Microbiology and Hygiene of the University of Montreal for gifts of normal and immune sera, to Dr. J. Gross for the protein fractions obtained from the Cutter Laboratories, Berkeley, Calif., to the Atlas Powder Co. (per G. F. Sterne & Son, Brantford, Ont.), for gifts of “Spans,” “Tweens,” “NNO,” and “BRIJ-30,” to the Canadian Industries, Ltd., Montreal, for gifts of “Elvanols,” and to Mrs. Mary Hersey for her able assistance in this work.

Using the paper chromatographic technic significant changes in the protein patterns have been demonstrated in patients with multiple sclerosis. We are unable to state at present the relationship of these changes to the activity of the disease, but our evidence would seem to indicate that they may occur concomitantly with, or immediately following the onset of exacerbation. Furthermore the changes in protein patterns revert to normal during remissions.

The binding of S35-labeled sulfate in rat serum has been investigated by filter paper electrophoresis. About 40% of the whole serum S35-sulfate activity was found to be combined with a serum constituent having the electrophoretic mobility of an α1-globulin. Although the chemical identity of the labeled substance has not yet been established, the results of these experiments suggest that it may be a protein with a sulfonated carbohydrate prosthetic group.

A method is presented for measuring Cortisol and corticosterone simultaneously in a single specimen of plasma. The steroids are purified by paper chromatography, eluted and measured by fluorometry. Losses incurred during analytical procedure are compensated by application of an isotope dilution correction. The normal range of corticosterone in human beings is 0 to 5 μtg/100 ml, with a mean of 1.3 ± 2.5 μg/100 ml. The normal range for Cortisol is 4.0 to 17.7 μg/100 ml, with a mean of 10.2 ± 3.6 μg/100 ml.

A log-probit graph paper is described by which a simple graphic estimation of the ED50and its standard error may be quickly made.

The use of blood plasma as a buffer for filter paper electrophoresis makes it possible to study the physico-chemical state of injected materials or of native blood constituents without subjecting the blood fractions to possible alterations by an artificial buffer. In veronal buffer yttrium appeared to exist in wholly immobile forms. In plasma buffer, yttrium existed in an immobile fraction and in an unionized fraction which moved with the solvent. Migration of materials as a result of solvent flow must be considered as a factor in the interpretation of electrophoretic studies of this type.