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RECENTLY, several workers1 have tried to fractionate gamma-globulins and immune sera by electrophoresis. Such experiments have only given the mean properties of all gamma-globulin proteins, since non-antibody as well as antibody proteins occur in immune sera. We have now carried out paper electrophoresis of a purified specific antibody.

IT has been shown by paper electrophoresis that xanthine dehydrogenase activity is bound to the globulin fractions of rat serum1. Using differential centrifugation, it was demonstrated that xanthine oxidase and xanthine dehydrogenase activities are present in the supernatant fluid corresponding to the ‘cell sap’ containing the soluble proteins of the rat liver2.

WE have studied a strain of Chaetomium globosum which was cultured on the basal medium of Buston and Basu1 (NaNO3, 2 gm.; KH2PO4, 1 gm.; KCl, 0.5 gm.; MgSO4.7H2O, 0.5 gm. ; FeSO4, 0.01 gm., in 1,000 ml. water) with the addition of 5 per cent sucrose and 2 per cent yeast extract. 50-ml. portions contained in 500-ml. flasks were inoculated with 1 ml. of a spore suspension and incubated at 25°. Samples were withdrawn aseptically from the culture flasks at intervals and spotted on a paper chromatogram2 and developed with n-butanol – water for 56 hr.

ON electropapergrams, the alpha-2-globulin protein peak has been shown to coincide exactly with the locus of maximum non-specific alkaline phosphatase activity1. This enzyme may be detected during the run by an adaptation of the Huggins and Talalay sodium phenolphthalein phosphate method, which determines alkaline phosphatase by ascertaining the colour due to free phenolphthalein produced at pH. 9.2 by incubating a serum sample with the colourless substrate (a blank correction is made for non-enzymatic substrate hydrolysis). Because of its broad optimum pH range, alkaline phosphatase has considerable activity at pH. 8.6, which is most often used for paper electrophoresis, and a significant proportion of free phenolphthalein is coloured at this pH. To detect alkaline phosphatase during paper electrophoresis, 0.1–1.0 per cent of sodium phenolphthalein phosphate is included in the buffer; the region of enzymatic activity is seen as a well-localized red band. At pH 9.2 the band is considerably more impressive in colour.

SEEGERS et al. 1 have very recently made an analysis on the amino-acid content, particularly tyrosine and tryptophan, of purified prothrombin. They have also shown by electrophoresis that their prothrombin preparation contains approximately 90 per cent of the protein in one component1. Several other workers2–4 have investigated the nature of the protein in prothrombin. Astrup and Darling4 have reported prothrombin to be a protein of globulin character. But the complete analysis of the prothrombin molecule with respect to different amino-acid contents is lacking.

THE monosaccharide composition of isolated cell walls of strain 7–11–05 of Chlorella pyrenoidosa Chick, an organism proposed for life-support systems in space travel, was examined. The two principal methods used were thin-layer chromatography and paper chromatography, accompanied by appropriate staining.

THE task of testing and localizing enzymes in a protein mixture subjected to paper electrophoresis is a tedious one. This is particularly true in a fractionation study where the number of tests may be numerous. During the course of such an investigation of the various carbohydrases in an Aspergillus niger fermentation, it became necessary to employ a rapid qualitative test for the detection of these enzymes. Keilin and Hartree1 have reported that glucose oxidase (notatin) can be successfully used for the quantitative assay of various carbohydrases. Monod and Torriani2 employed notatin to determine glucose in their studies of amylomaltase. Recently, Whistler et al. 3 showed that D-glucose in corn syrups can also be quantitatively estimated by using notatin. The latter enzyme oxidizes glucose to yield two products—gluconic acid and hydrogen peroxide.

THE study of animal haemoglobin types is developing rapidly and the results obtained in various species have recently been summarized by Huisman, van der Helm, Visser and van Vliet1. However, the electrophoretic mobility of hippopotamus haemoglobin does not seem to have been studied before, possibly because of scarcity of materials. We have had the opportunity to examine haemoglobin samples from the East African hippopotamus, Hippopotamus amphibius L.2, obtained during a controlled reduction in a hippopotamus population.

PAPER-PARTITION chromatography of tissues of genetically known strains of Drosophila melanogaster as well as those of certain plants has shown that each strain gives a constant and distinctive pattern. The chromatographic patterns of heterozygotes carrying recessive genes can be easily distinguished from those of either parental homozygous strain, even though the phenotype of the heterozygote is indistinguishable from that of the homozygous dominant. This is the case for: (a) the fluorescent patterns observed under ultra-violet light in the chromatograms of adult Drosophila melanogaster heterozygous for the following mutants: bent, eyeless2, short-vein, spineless-aristapedia, vestigial; (b) the ultra-violet-fluorescent and the ninhydrin-positive patterns of root-tip smears of heterozygotes for the mutant male-sterile2 in tomato1; (c) the ultra-violet-fluorescent and the ninhydrin-positive patterns of root-tip smears and leaf extracts of heterozygotes for the yellow-green mutant of musk melon2 (see also chromatogram).

ELECTROPHORETIC fractionation of pancreatic juice components in a dog has been described in the literature1–4. Some of the authors2,3 isolated part of the fractions and studied their enzymatic activities. All authors used the free electrophoresis method with the exception of Delcourt and Delcourt4, who employed paper electrophoresis for the separation of the different fractions.

THE use of 'Gammexane' has been recommended for controlling insect pests in libraries and record offices, where insect infestation is a serious problem especially in a tropical climate. 'Gammexane' smoke generators appeared to be particularly suitable for this purpose owing to the ease and safety with which they can be handled. But no information about its effect on paper1 was available, and the use of a new insecticide for the disinfestation of documents of a unique and irreplaceable nature had therefore to be scrutinized from the preservation point of view.

XANTHINE dehydrogenase from Drosophila melanogaster is of particular interest since mutants at two separate loci lack this enzyme1 while a mutant at a third locus produces about 25 per cent of normal activity2. In an effort to detect strains of flies with genetically altered forms of xanthine dehydrogenase we devised a technique involving paper-strip electrophoresis in which this enzyme is located after electrophoresis by spraying the strip with the enzyme's substrates, incubating it at 25° C, and then using paper chromatography to separate the fluorescent product from the substrate which also fluoresces. This technique is very simple, and has potential application to other enzymes. Paper electrophoresis of xanthine dehydrogenase in rat serum has previously been reported using the technique of elution and direct assay3,4.

WE have already described a fortuitous combination of events which led to the discovery that American newspapers and other paper products contain a potent analogue of the juvenile hormone of the European bug, Pyrrhocoris apterus L. (Heteroptera ; Pyrrhocoridae)1. When reared in contact with ‘active paper’ or when exposed to surfaces impregnated with extracts of the ‘paper factor’ (PF), fifth-stage Pyrrhocoris larvae undergo one or more supernumerary larval moults and finally die without completing metamorphosis or attaining sexual maturity. This same result is observed when 1 µg of partially purified PF is topically applied to young fifth instar larvae1,2.

IN the first report1,2, on ‘crossing paper electrophoresis’, a direct demonstration of enzyme-substrate complex has been described. The enzymes tested included crystallized pure trypsin, chymotrypsin and ribonuclease.

IN several recent publications, separation of the alkaloids of the South American Strychnos into a great number of constituents by paper chromatography has been recorded1,2. The most successful solvents for two-dimensional analysis yield, however, almost the same sequence of spots. Thus it was considered desirable to find methods of separation which gave sequences different from those obtained by partition chromatography.

IN a communication under the above title, Giri1 describes the use of a starch agar gel for the location of amylases on the paper chromatogram. The technique seems to be essentially the same as that used in this Department, and previously described in Nature2.

IN connexion with the chromatographic separation of alkaloids of Chelidonium maius L.1, I found alkaloids giving rather big spots with a tendency to form tails. This is connected, among other things, with the dissociation constant (K B), the value of which influences the course of the chromatographic process, organic electrolytes having different values of K B in the water phase and the organic phase of dissociation.

IN the course of studies on the aromatic constituents of Theobroma cacao L., a method has been developed for the separation by one- and two-dimensional paper chromatography of the naturally occurring substituted cinnamic acids, which is also applicable to many other aromatic acids of plant tissue.

‘QUICK decline’, a devastating virus infection of orange trees, was first found to be present in southern California in 1939, and by 1952 it had spread to six of the fourteen counties in which oranges are grown in California. Inoculation tests1 demonstrated that tolerance to the virus varies with the variety of the rootstock on which the sweet orange is growing, and that the disease is likely to be fatal only if the rootstock is that of the sour orange (Citrus aurantium L.). Because of this scion-rootstock relationship to the seriousness of the disease, which in one form or another is widely spread throughout the citrus-growing areas of the world, a method for the detection of the sour orange rootstock in established orchards is of considerable significance.

EVIDENCE for the presence of growth substances in the root extract of water hyacinth (Eichhornia speciosa Kunth), presumably related to the rapid multiplication of the weed, has been reported previously from this laboratory1. A comparison of the effects of the extract with those of indole acetic acid or gibberellic acid indicated a different substance or substances present in the extract.

POLYCHLOROBIPHENYLS (PCB) cause chloracne and liver damage in man and induce oestradiol degradation1. They are widely distributed in the ecosystem in birds, fish and shellfish1–4. The miserable episode of “Yusho”, a mass food poisoning involving more than 1,000 people which was caused by chronic ingestion of rice oil contaminated with ‘Kanechlor-400’, a commercial brand of PCB, occurred in western Japan in 19685,6.

REPORTS have been published describing increases in growth and vigour of pasture in New Zealand following the experimental transplantation of lumbricid earthworms of the species Allolobophora caliginosa into soils where they were not normally found1–3. Fourteen species of Lumbricidae are known in New Zealand, although the family is not native to the region4. Since they have been introduced accidentally during the past hundred years or so there has not been sufficient time for them to spread by natural means to all areas under grass. The increased pasture yields may be due to the presence of plant growth-promoting compounds elaborated by earthworms and secreted by them into their casts and thus into the soil. A similar suggestion was, in fact, advanced by Hopp and Slater5, who found in pot experiments that the addition of both living and dead earthworms was associated with increased growth of the test plants.

THE acid phosphatase test has proved its usefulness in seminal stain examinations and is now in widespread use1–4. The desirability of preparing a stable form of the reagent was obvious, more especially for ‘scene-of-crime’ searching, post-mortem examinations and for scientists and clinicians without laboratory facilities.

ADVANCES in auxin physiology within recent years have been limited by continued uncertainty as to the identity of the auxins present in various plant tissues, and by the absence of reliable methods of separating the growth-promoting and the growth-inhibiting substances present in plant extracts. The technique of paper chromatography, used in conjunction with biological tests, would seem to offer a ready solution to both these problems.

MOST methods used for the estimation of strychnine in nux vomica lead to gross inaccuracies due to brucine interference. Even oxidation of brucine with nitric acid under optimal condition (5 min. at 60° C.) leads to errors due to partial destruction of strychnine1. Gravimetric estimations also require amounts greater than 0.1 gm. in the sample1.

A METHOD for using paper partition chromatography for taxonomic studies on land snails has been described by Kirk et al. 1.

WE have examined recently blood containing fibrinolysins in high titre. The blood was taken from a man who was later diagnosed as suffering from prostatic malignancy. The presence of high-titre fibrinolysins, raised formal acid phosphatase, and greatly reduced fibrinogen content, were all carried out by routine, accepted biochemical methods, and were fully diagnostic. It was then considered that there could be present a circulating auto-immunological factor which we might be able to demonstrate.

IT has been shown1 that, by the action of chymotrypsin, insulin is broken down into a number of peptide fragments of comparatively small molecular weight, and a more massive residue. The higher molecular weight material can be precipitated by means of trichloro-acetic acid. Attempts have been made to separate the lighter peptide mixture, and the most effective method so far found has been paper partition chromatography2.

CHROMIUM sesquioxide (Cr2O3) is widely used for estimating feed digestibility1 or faeces output2 by ruminants, but such estimates may be subject to considerable errors because the concentration of this marker in faeces normally shows considerable diurnal variations even after prolonged regular administration. These variations are particularly obtrusive with grazing animals where the chromium sesquioxide, in this case used for estimating the output of faeces, is usually administered in concentrated powder form in gelatin capsules once or twice daily, and they probably arise primarily from an imperfect mixing of the oxide with digesta in the reticulo-rumen3,4. This view is supported by unpublished observations (by J. F. D. Greenhalgh) on the concentrations of chromium sesquioxide in the organic matter of digesta obtained from the duodenum of a sheep permanently cannulated at this point. As the variations in concentration during a period of regular dosing showed the same pattern and were greater than those found in the faeces, it was decided to study in a comparably cannulated sheep the passage of single doses of oxide powder, each given in a gelatin capsule and in amount large enough to give measurable concentrations over a considerable period of time. Samples of digesta were drawn from the duodenum at intervals beginning 1 hr. after dosing. Results from a typical run where 8 gm. were given are shown in Fig. 1 (line A) and indicate that a large part of the dose had passed from the stomachs within 4 hr. of administration. The probable explanation is that the capsule container dissolved quickly in the anterior rumen or reticulum3 and that local concentrations of chromium sesquioxide in these regions4 were rapidly transferred through the reticulo-omasal orifice.

MATERIALS composed of American paper-pulp have been shown to contain an extractable, heat-stable lipid which exhibits high juvenile hormone activity when topically applied or otherwise brought into contact with the European bug, Pyrrhocoris apterus L. (Heteroptera; Pyrrhocoridae1,2). The active principle is synthesized by certain species of pulp trees—more particularly, the American balsam fir (Abies balsamea). The ‘paper factor’ (PF) proves to be an extremely potent analogue of the juvenile hormone of Pyrrhocoris apterus3. Yet, so far, it has proved completely inactive when tested on other laboratory insects including two other species of Heteroptera—Oncopeltus fasciatus (Lygaeidae) and Rhodnius prolixus (Reduviidae)1,2.

SEVERAL years ago, Gibbs,1 summarizing contributions of biochemistry to plant taxonomy, recognized the importance of the then relatively new techniques of paper chromatography and forecast their wide application, to systematics. Although a number of workers have applied chromatographic techniques to taxonomic problems in both botany and zoology2, the use of these techniques is not nearly commensurate with their potential value, perhaps because the cooperation of taxonomists and persons trained in biochemical analysis has been slow to develop. The present communication constitutes a brief description of work currently under way which is to be reported in greater detail elsewhere;3 the chomatographic analysis of individuals from a natural hybrid swarm of Baptisia laevicaulis × B. viridis (family Leguminosae), and correlation of these data with a similar analysis of the parental species collected in pure populations. Three other species of Baptisia were also examined chromatographically. Since this genus contains about 30 species of wide occurrence in the eastern United States, and hybridization among these species is common, it is highly probable that the scope of the present investigation will be extended. In this study we were particularly concerned with the recombination of biochemical components peculiar to each parental species among the hybrid and back-crossed individuals.

THE early stages in the life-history of recent crinoids have always been regarded with interest, because it was hoped that they would throw light on the evolution of this class, so rich and various in ancient seas, and on its relation to the other very differently fashioned classes of Echinoderma. Unfortunately, the only forms that have up till now furnished material for the em-bryologist are the unstalked comatulids, or feather-stars, and in the past such material has come from but a single genus, and from only three closely allied species of it—Antedon bifida of our own coasts, A. mediterranea, and A. adriatica. The accounts of their development by W. B. Carpenter, Bury, Seeliger, and others have shown slight differences, due, in part, probably to specific distinctness of the material. Even if it were not feasible to obtain the early stages of any stalked crinoid, still a study of other species, representing other genera of comatulids, was much to be desired, since it might then be possible to infer which features were peculiar to Antedon and which were common to comatulids generally, if not to the whole class Crinoidea. Such a study has now been made by Dr. Morten-sen, who has obtained a fairly complete series in four genera, and the pentacrinoid larvae of two others. His results are set forth in clear English with his usual care, and the memoir is illustrated by admirable drawings from his own pencil. His many interesting results are discussed in a “General Part” which demands the attention of professed morphologists. Here we shall select for comment a few observations that bear on the past history of the class. Papers from the Department of Marine Biology of the Carnegie Institution of Washington. Vol. xvi. Studies in the Development of Crinoids. By Th. Mortensen. (Publication No. 294.) Pp. v + 94+xxviii plates. (Washington: The Carnegie Institution of Washington, 1920.) 6 dollars, post free.

THE existence in human and animal serum of protein constituents which migrate during electrophoresis at rates faster than that of albumin has been widely reported in the literature by investigators using moving boundary electrophoresis1 and electrophoresis on paper2–5 and in hydrolysed starch-gels6,7. Using tris buffer (pH 8.9), which consists of tris-(hydroxymethyl)aminomethane, ethylenediamine tetraacetic acid (EDTA) and boric acid, Aronsson and Gronwall8 reported not only an enhanced separation of the protein constituents of human serum and the appearance of a pre-albumin component, but also a partial splitting of the albumin peak during electrophoresis in tris/boric acid buffers.

IN the course of our investigations of Echinocystis lobata seeds, it became desirable to locate triterpenoid glycosides on paper chromatograms by means of a colour reaction. A modification of the Liebermann–Burchard test described by Neher and Wettstein1 was unsatisfactory for this purpose. We found that the original Liebermann–Burchard reaction applied to paper chromatograms with comparatively little alterations gave more satisfactory results, permitting a reliable detection of the spots.

Biochemical Differences between Inbred and Outbred lines of Drosophila melanogaster studied by Paper Partition Chromatography

ON reading the article entitled “Juvenile Hormone Activity for the Bug, Pyrrhocoris apterus”, by Slama and Williams1, I was struck by a likely correlation.

DURING the course of an investigation into the chemistry of the cell wall of Penicillium notatum an unusual amino sugar was detected in acid hydrolysates of the walls. It was present in only small amounts, but was easily visible by paper chromatography. The material was isolated from a 4 normal hydrochloric acid hydrolysate (carried out at 100° C for 24 h) and was purified by elution from Whatman 3 MM paper using solvent system B. It gave a positive reaction with the silver nitrate1, aniline phosphate2, ninhydrin and Elson–Morgan3 sprays, suggesting that the compound is probably a 2-deoxy, 2-amino sugar.

PAPER electrophoresis became a suitable technique for embryological purposes when Kohn1 proposed the use of pure cellulose acetate as a supporting medium instead of filter paper. This method enables us to separate much smaller amounts of protein than hitherto : one single amphibian embryo yields enough protein to give a clear electrophoretic pattern.

RECENTLY, several articles have been published on the multiplicity of cellulase. Using paper chromatography Jermyn1 found eight components in cellulase from Aspergillus oryzae, each with cellulolytic activity. Reese and Gilligan2 found, also with chromatographic techniques, three components in Myrothecium cellulase, whereas Miller and Blum3, using starch electrophoresis, found at least eight components in this product. Grimes4 isolated from Myrothecium cellulase six components, of which possibly three had cellulolytic activity, and Thomas5 found seven protein fractions by paper electrophoresis. These facts are puzzling. Whitaker6 succeeded in isolating a homogeneous protein from crude Myrothecium cellulase which exhibited all the activities of the crude product, so there is a possibility that cellulase is a single enzyme. However, this has never been confirmed.

IT has been reported previously1 that the short-day plant, Maryland Mammoth tobacco, will flower under long day-lengths (14½–15½ hr.) if exposed to varying dosages of γ-rays. Recently, we have been investigating the effect of γ-radiation on the endogenous level of the indole compounds occurring naturally in leaf, stem, root and apical tissues of γ-irradiated and normal Maryland Mammoth tobacco. Large quantities of these tissues (300–1,000 gm.) have been extracted with absolute ethanol, employing a technique for extraction and paper chromatography as described previously2. Microlitre quantities of the extracts, after acidification and extraction with ethyl ether, were spotted on Whatman No. 1 filter paper and chromatographed in seventeen different solvents. Colours were developed with p-dimethylaminobenzaldehyde. 3-Indoleacetic acid was not detected in any of more than thirty-five individual extractions of leaf, stem, root or apical tissues of normal or γ-irradiated Maryland Mammoth tobacco. However, an unidentified indole compound was found in the leaf and apical extracts with R F values as shown in Table 1.

ISOLATED chloroplast preparations offer great advantages in the analysis of carbon pathways of photosynthesis because the system is not complicated by non-chloroplast metabolism, and kinetic analyses1 can be made which are difficult with whole leaves2. Unfortunately the slow diffusion and equilibration of CO2, HCO−3 and CO2−3 in the suspension medium is a serious disadvantage in gas exchange or 14CO2-12CO2 pulse-chase experiments3, but this difficulty can be overcome by using a thin film of concentrated chloroplast suspension spread on a non-absorbent support in the form of an “artificial leaf”. Recent experiments by Coombs and Baldry have shown that pea and spinach chloroplast preparations supported on filter paper were stable only for periods of up to 10 min4. We have used Acetabularia mediterranea Chloroplasts on filter paper to analyse the products of short term (1 s to 5 min) 14CO2 fixation, but we found them insufficiently stable for longer experiments or gas exchange measurements. We have overcome this difficulty, however, by supporting Chloroplasts on a non-absorbent grid instead of filter paper.

Simple oligonucleotide maps can be obtained by two-dimensional paper electrophoresis from enzyme digests of 5S RNA from KB carcinoma ribosomes. These maps can be distinguished from those obtained from 5S RNA derived from E. coli and from RNA components of low molecular weight derived from KB cells with and without adenovirus infection.

THE antigenic properties of insulin are now well recognized, but no simple method has yet been described for the routine detection and assay of insulin antibodies. The method described here is based on the classical observation by Berson, Yalow, Bauman, Rothschild and Newerly1. They showed that during chromato-electrophoresis on filter paper, 131I-labelled insulin bound by insulin antibodies in human serum moves with the β-γ-globulins, while free insulin remains fixed at the point of application of the serum-insulin mixture on the filter paper. When an excess of unlabelled insulin mixed with a trace of labelled hormone is added to the anti-insulin serum, the excess insulin is adsorbed from solution by cellulose and the amount bound by the antibodies is measured in the supernatant solution. We have used this assay system for serum obtained from guinea-pigs treated with bovine insulin2 and capable of neutralizing insulin at rates ranging from 0.1 to 3.5 units per ml. serum. It can, however, be adapted for the detection and assay of antibody concentrations lower than this; it has not yet been used for anti-insulin sera of other animals or of man.

ANTIMONY pentachloride has been used as an agent for the detection of steroids in paper chromatography by a number of investigators1–4. It has been found that antimony pentachloride is a useful agent for detecting certain digitalis compounds (digoxin, digitoxin and digitoxigenin) on paper chromatograms.

Detection by paper electrophoresis. Using starch gel electrophoresis, six cattle1, fourteen sheep2, three goat (see later) and three human3,4 β-globulin phenotypes have been recognized and indicated to be under the genetic control of three, five, two and three allelomorphs respectively. As one of us had noticed a variation in the number and position of the β-globulin zones in paper electrophoresis of animal sera, the two methods have been compared on sera from cattle, sheep and goats. Starch gel electrophoresis was carried out in phosphate buffer1 pH. 7.6, paper electrophoresis on a wide strip of horizontally suspended Whatman 3MM paper in barbiturate buffer pH 8.6, I 0.05. Typical examples of corresponding electropherograms of sheep and cattle sera are shown in Figs. 1 and 2. The electropherograms of goat sera shown in Fig. 3 are discussed below.

USING paper chromatographic methods it has been shown that, following digitoxin administration to rats, the urine contains small amounts of unchanged digitoxin together with larger quantities of two cardioactive metabolites previously designated metabolites C and G 1,2. Human urine collected for 12 hr. from normal adults after an oral dose of 1 mgm. of digitoxin has now been found to contain some unchanged digitoxin as well as apparently larger quantities of two metabolites. The two metabolites when eluted from paper and rechromatographed using two systems of paper chromatography could not be separated from metabolites C and G.

VARIOUS β-globulin types have been demonstrated, using starch-gel electrophoresis, in several species of mammals, namely, man1, cattle2, sheep3, goat4 and horse5. The present communication reports the discovery of similar variations in two species of monkey, Macaca mulatta and M. irus. Variations in the monkey β-globulins were initially detected by paper electrophoresis using barbital buffer at pH 8.6. However, the β-globulins were much better resolved when starch-gel was used instead of paper.