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1 A new diagonal electrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ɛ-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2 The technique has been successfully tested with insulin. 3 When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-Tyr-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The three basic amino acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4 A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequences of the two proteins.

1. A new method is described for f∈≥rpr∫∈g'cysteicaceptsderivedomthedisϕ––debrsofprote∈s.Cyst∈epeptsareseparatedbypapere≤ctrophoresisand⊗zedonpaperbyperformicacapour.E≤ctrophoresisatright∠s→thefirstdirection∏ucesparal≤lgroupsofcysteicaceptsly∈goffadiagonal.Thisf∈≥rpr∫∈g′cysteicaceptsderivedomthedisϕdebrsofprote∈s.Cyst∈epeptsareseparatedbypapere≤ctrophoresisand⊗zedonpaperbyperformicacapour.E≤ctrophoresisatright∠s→thefirstdirection∏ucesparal≤lgroupsofcysteicaceptsly∈goffadiagonal.Thisfingerprint' reveals the way in which the cysteic acid peptides were originally joined in the protein. 2. The method allows a very easy selective purification of cysteic acid peptides. 3. By applying this method to bovine chymotrypsinogen A, we found that the half-cystine residues were linked 1–122, 42–58, 136–201, 168–182 and 191–220.

In vitro dry matter disappearances were determined for 11 paper sources and three complete rations containing 0, 10, and 20% newsprint. Inoculum was from an alfalfa hay-fed steer. Alfalfa hay, brome hay, and Solka Floc were reference substrates. Brown wrapping paper and brown cardboard had the highest dry matter disappearances of 90.8 and 77.8%; whereas four glossy or slick magazine papers had significantly lower values of 46.1, 45.1, 41.3, and 41.0%. Newsprint with black ink, without ink, and with colored inks had dry matter disappearances of 33.2, 32.6, and 26.5%, which were significantly lower than those of the glossy magazine papers. Lowest dry matter disappearances of 24.0 and 20.1% were from two magazine papers. The complete rations had 77.9, 80.1, and 81.8% dry matter disappearances for the 0, 10 and 20% paper rations. These data indicate substantial differences in digestibilities of various paper sources.

A simple technique is described for drying sera on washed, dry sand or ordinary sugar cubes for the sero-diagnosis of leptospirosis. The results are shown to be very similar to those obtained with fluid sera or sera dried on filter paper discs. Sera adsorbed on sand or absorbed in sugar eliminated some of the problems associated with sera dried on paper. This method is suitable for use in the field and is expected to be of value in epizootiological studies where contamination and chemical denaturation of fluid serum samples held without refrigeration is a problem.

The purpose of this study was to isolate and characterize major 14 C-labeled compounds excreted in urine of an ovariectomized heifer following intravenous injections of 4- 14 C-corticosterone (4.96 μ Ci) and 4- 14 C-cortisoI (4.28 μ Ci). Only 7.4% and 11.7% of the injected radioactivity were recovered in urine during the first 12 hours after injection of corticosterone and Cortisol, respectively, and 53% and 62% of this urinary radioactivity were extracted. Enzymatic hydrolysis and sol-volysis procedures were used in sequence. The urine was extracted with ethyl acetate before and after each hydrolysis. Isolation of distinct zones of radioactivity was accomplished by column (one system) and paper chromatography (three systems). Partial identifications of labeled compounds were made by matching their mobilities to those of authentic standards during paper chromatography. Ultraviolet light, blue tetrazolium, and phosphomolybdic acid were used as indicators of certain reactive groups. Due to small quantities of labeled metabolites only tentative identifications were possible. Isolations corresponding to standards of 6-hydroxycortisol represented 35% of the radioactivity recovered after injection of 14 C-cortisol as compared to 19% tetrahydrocortisol, 6.4% cortols, 4.5% cortolones, and 1.6% tetrahydrocortico-sterone. Similarly, for 14 C-corticosterone, 34% of the radioactivity recovered and isolated was from an unknown highly polar compound(s) as compared to 17.6% tetra-hydrocorticosterone, 15.7% tetrahydrocortisol, 3.6% 6-hydroxycortisols, 2.4% cortolones and 2.2% cortols.

The primary structure of sheep para-xA-casein, the N-terminal portion of x-casein, was established. The reduced alkylated protein was subjected to digestion with trypsin. The resulting soluble peptides were purified by a combination of Dowex 1 X2 chromatography and electrophoresis and chromatography on paper; the core peptides were solubilized in 50% formic acid and filtered on Sephadex G-50. The amino-acid sequence of these peptides was determined chiefly with a Sequencer. Alignment of the tryptic peptides into a single chain containing 105 amino acids was determined from basic overlap peptides (tryptic core peptides with several basic amino acids; chymotryptic peptides). Some comparisons were achieved with cow para-xa-casein. %-Casein is the principal casein fraction affected by chymosin (or rennin) in the primary phase of the milk clotting process [ 11. The group of Jollbs established already in 1965 that during this enzymic reaction, a Phe -+ Met linkage was specifically split in cow as well as in sheep x-caseins [2-41. An insoluble part, para-x-casein (N-terminal moiety of x-casein), was from Merck or Prolabo except those employed for the Sequencer which were purchased from Socosi (94100Saint Maur, France) and 4-sulfophenylisothiocyanate from Pierce.

1. Details of an improved method for starch-gel electrophoresis of water-soluble muscle proteins are given. 2. Methods are described for detecting enzyme activities on the starch gel after electrophoresis, by using pieces of filter paper. 3. Compositions of incubation mixtures suitable for detecting any of the enzymes of glycolysis, and certain other enzymes, are given. 4. A comparison of the various enzymes in extracts of several muscles from one rabbit was made; most differences are quantitative only. 5. A detailed comparison of the mobilities of various enzymes in extracts of muscles from a wide variety of species was made. Each species was found to have a characteristic pattern of proteins on the starch gel, and the mobilities of individual enzymes varied considerably. 6. Potential uses and extensions of the methods are discussed.

The Achilles tendon is built up mainly of collagen and it contains minute amounts of ground substances and cell materials. Opinions about its content of polysaccharides vary considerably. The ground substance of connective tissue is rich in acid mucopolysaccharides and Meyer (1957) has isolated the chondroitin sulphates from Achilles tendon. Besides the acid mucopolysaccharides the presence of other mucopolysaccharides has been suggested. First Grassmann & Schleich (1935) showed that collagen isolated from the skin and from Achilles tendon contains 0'5-0 7 % of polysaccharides. Consden (1953) and Consden, Glynn & Stanier (1953), using a combination of paper electrophoresis and paper chromatography, have shown that the connective tissues contain polysaccharides which after acid hydrolysis yield glucose, mannose, galactose and glucosamine. Bowes, Elliott & Moss (1955), and especially Moss (1955), are of the opinion that the negligible amount of hexosamine and the very small amount of hexose found in collagen may be impurities. With the aid ofthe enzyme collagemnucoproteinase isolated from the pancreas (Banga & Balo, 1956) the present authors succeeded in showing the existence of a protein fraction, collagenmucoprotein2 (mucoid2), the polysaccharide component of which, it was suggested, was not identical with the acid polysaccharides (Banga, 1958). It was thought that this substance might be responsible for the periodic acid Schiff reaction given by connective tissue. Mucoid2 is one of the important constituents of collagen fibre (Banga, 1958). It also takes part in the chemical contraction relaxation phenomenon of collagen (Balo, Szabo & Banga, unpublished work). In the present paper the isolation and analysis of mucoid2 are described.

1. UDP-glucose dehydrogenase (EC 1.1.1.22) was extracted from epiphysial-plate cartilage of newborn pigs and from whole bovine corneas. 2. Formation of UDP-glucuronic acid was demonstrated by radioautography after separation of the sugar nucleotides by paper chromatography or t.l.c.: in these conditions a radioactive glucuronic acid spot also appears. 3. UDP-xylose prevented the formation in the incubation mixture of both UDP-glucuronic acid and free glucuronic acid. 4. In both tissues the dependence of the enzyme activity on pH and the Km values for UDP-glucose and NAD+ were determined. 5. Inhibition by UDP-xylose with respect to UDP-glucose was investigated. The plots of 1/v versus 1/[UDP-glucose], and of percentage inhibition versus UDP-xylose concentration and the Hill coefficient showed that a co-operative effect existed between UDP-xylose-binding sites. 6. The physiological meaning of the different affinities of cartilage and cornea enzymes for UDP-xylose is discussed and related to the different glycosaminoglycan contents of the two connective tissues studied.

The experiments reported here were carried out to study the in vitro binding of 131 I to bovine milk protein and the factors influencing this binding. Incubation of inorganic carrier-free 131 I-iodide with fresh raw milk at 37C for two hours showed as much as 50% of the 131 I bound to milk protein, and that as much as 80–90% could be bound in 12–24 hours. It was also shown that this reaction is both time- and temperature-dependent and could be readily blocked by incubation at 8C, boiling the milk prior to incubation, addition of carrier iodide or antithyroid agents such as Tapazole (methyl mercaptan imidazole). Paper chromatography of enzymatic digests of the milk showed the protein-bound 131 I in milk is present as 131 I-labeled monoiodotyrosine. No other 131 I-labeled compounds were observed. These studies would tend to indicate that the binding of 131 I-iodide to milk protein involves an enzymatic oxidation of the iodide.

Fifteen lactating Holstein cows in a 3×3 Latin square experiment determined effects of ground newspaper in the diet. Test components of the rations were: A) 20% cottonseed hulls; B) 10% paper and 10% cottonseed hulls; C) 20% paper. All animals received 2.3kg of long alfalfa hay daily. Proximate analysis and gross energy determinations indicated that the paper rations contained more crude fiber, ether extract, and gross energy and less crude protein and ash. Ink in the paper apparently caused higher gross energy values and probably increased ether extract. Estimates of dry matter digestibility by an in vitro technique were not significantly different being 77.4, 77.4, and 77.5 for the control, 10% paper, and 20% paper rations. Average daily ration intakes were significantly different among rations with intakes of 21.5, 17.0, and 12.5kg per day for the control, 10% paper, and 20% paper. Paper at 20% of the ration significantly lowered actual milk yields but did not significantly lower fat-corrected milk yield during the six weeks. Average daily fat-corrected milk yields for control, 10% paper, and 20% paper treatments were 15.9, 16.9, and 16.3kg. Milk fat percentages were 2.6, 3.1, and 3.4 with significant differences between treatments. Milk protein and solids-not-fat were not significantly different; however, there was an inverse linear response between treatment and milk protein percentage. There were small differences in ruminal volatile fatty acids with a significantly lower pH for the 20% paper compared with the control. Paper at 10% in the ration would maintain milk fat percentage without reducing actual milk production during six weeks.

Two unknown radioactive areas appeared after radioautography and two dimensional paper chromatography of culture medium in which Escherichia coli was grown. These materials were studied by paper chromatography and paper electrophoresis of several derivatives and identified as ethionine and ethionine sulfone, the latter an artifact. Chromatographic coincidence of the unknowns and their derivatives with authentic materials establishes the identification. Ethionine was found in cellular extracts and in the growth media of Escherichia coli, Bacillus megaterium, Pseudomonas aeruginosa, and Aerobacter aerogenes but not in Scenedesmus, Saccharomyces cerevisiae, or bovine lymphosarcoma cells. Ethionine was synthesized by resting E. coli cultures from radioactive sulfate and from radioactive methionine. Growing cells labeled ethionine within 1 minute after addition of radioactive sulfate to cultures. Levels of radioactivity in ethionine increased with time. No incorporation of this amino acid could be detected in the cellular proteins formed under the conditions of this study.

When the alkaline fast green reaction is applied to calf thymus histone fractions f 1, f2a, f2b and f3 on Millipore filter paper it is found to stain equal amounts of these fractions equally well. The dye reaction, therefore, seems to depend on the over-all charge and not exclusively on the per cent arginine plus lysine in the histone fractions. This staining reaction has been applied to histone fractions separated by acrylamide gel disc electrophoresis. Deamination of the histone fractions on both paper and acrylamide gel resulted in a reduction in the intensity of the stain. The stain that remained followed the relative amount of arginine in each fraction or band. In the gels, the fi band was removed completely. This confirms previous interpretations of the cytochemical deamination reaction. The stain reactions have been utilized as a means of comparing electrophoretically resolved histone bands of similar mobility from crickets and mealy bugs. Simice Alfert amid Geschwind (2) developed the alkaline fast greeui staiuiimig reaction it has become omie of the miiore PoPular methods for the cytochemical localization amid analysis of basic nuclear proteitis. Although its precise chemical mechanism is not umiderstood, the stainiuig is thought to depemid omi the miet positive charge of the protein molecule at high pH (2). In histones, the s-amino group of lysine and the guanido group of arginine (11) are priniarily responsible for the dye binding. Usitsg the alkaliuie fast green reaction with and without the Van Slyke deamination, Bloch and Hew (5) demonstrated a tramisit ion in the arginine arid lysine content of histones during sperrniogenesis. Other authors have continued to use this

During purification of D-erythrulose ruductase, the existence of a protecting factor which prevents the enzyme from cold inactivation was confirmed. The factor was purified by ion exchange and paper chromatography and was identified as NADP+ by paper chromatography, paper electrophoresis, determination of the components, and spectroscopic properties.

We have further characterized the polynucleotide ligase purified from cultures of the human heteroploid line EUE [Spadari, Ciarrocchi and Falaschi, Eur. J. Biochem. 22, 75 (1971)]. The 350-fold purified enzyme gives positive response with four different assays; the rates with the different substrates are different, but the variations are identical to those observed with the purified ligase induced by T4-phage. The enzyme can reconstitute the transforming activity of Bacillus subtilis DNA inactivated by pancreatic DNAase. The human cell enzyme is unable to use a hybrid substrate where an interrupted polydeoxynucleotide is annealed to a polyribonucleotide, whereas in the same conditions the T4 enzyme gives appreciable activity. The partial dependence of the purified enzyme on proteins present in a boiled crude extract, reported in the previous paper, seems due to an aspecific protective effect of the soluble proteins of cell extracts. The enzyme does not show any appreciable sequence specificity in the phosphodiester bond it can form. The purified enzyme can be fractionated into two molecular forms, one having a molecular weight of 190000, the other 95000; fresh extracts of EUE cells contain almost exclusively the high molecular-weight form; ageing of the extract or purification lead to the appearance of the second peak, without variations in the total activity. This could correspond to the conversion of a dimer structure into a monomer. The “dimer” has a pH optimum close to 8.1, whereas the “monomer” has its optimum at 7.5; this explains the bimodal pH curve previously described in the purified enzyme.

1. Proteinuria was found in most sucking lambs and calves examined but was less prevalent in bottle-fed kids or bucket-fed calves. 2. This incidence of proteinuria corresponded to the presence of immune lactoglobulin in the serum. 3. The extent of proteinuria in lambs and calves rose to 20 g./l. or more, whereas in kids values of only 4 g./l. were obtained. 4. The proteins present in the urine of lambs and kids have been compared with those in the urine of calves by using boundary electrophoresis and paper electrophoresis. 5. Sedimentation analyses have been made on the proteins of lamb, kid and calf urine. Components with sedimentation coefficients 3s and less were present. The separated slow electrophoretic components were found to have sedimentation coefficients 3·3, 3·6 and 3·3s respectively. 6. The sedimentation analyses, and for calf urine the electrophoretic data, support the view that the slow-moving electrophoretic components of calf and lamb urine are degraded immune lactoglobulins. 7. There was electrophoretic evidence for other degraded proteins in some lamb urine which showed multiple zones that could not be related to serum or colostral proteins.

The amino acid sequence of γ-crystallin (fraction II) from calf lens has been determined; this indicates it to be a single-chain polypeptide of 165 amino acid residues.

The nature of the carbohydrate-protein linkage in the chondroitin 4-sulfate-protein complex of bovine nasal septum has been investigated. Two carbohydrate-serine compounds, xylosylserine and galactosylxylosylserine, were isolated from the complex by enzymatic degradation followed by mild acid hydrolysis. Their structures were established as O-β-d-xylopyranosyl-l-serine and 4-O-β-d-galactopyranosyl-O-β-d-xylopyranosyl-l-serine. These findings show that the carbohydrate-protein linkage in the chondroitin 4-sulfate-protein complex is a glycosidic linkage between xylose and the hydroxyl group of serine.

1. Cysteic acid peptides from various digests of calf rennin were purified by diagonal paper electrophoresis. 2. The amino acid sequence of these peptides accounts for 38 amino acids around three unique disulphide bridges in rennin. 3. One bridge connects two acidic regions of the chain, one forms a loop of five residues and the other a loop of six residues. 4. These bridges are homologous with those of hog pepsin. 5. Tryptic peptides from the C-terminus of rennin account for 22 residues, 17 of which are homologous with the C-terminus of pepsin. 6. Altogether, sequences accounting for 94 of the 270 residues in rennin are described and the degree of homology with pepsin approximates to 70%.

The secretion of citric acid by the bovine female genital epithelium has been investigated by Gaspar & Cseh (1963). In 60% of cases no citric acid was present, but in the secreta of 40 % of the cows, quantities corresponding to spots of 1 \m=.\5to 40 \g=m\gcould be demonstrated. In 30 % of the investigated cases one unidentified spot, usually accompanied by citric acid and showing Rf values from 0\m=.\78to 0\m=.\80,also appeared on the chromatogram. We have been trying to identify more closely the compound forming this spot. It was diffusible and remained unhydrolysed in 1 n-sulphuric acid for 2 hr and was in character acid. Although the unknown compound was generally accompanied by citric acid, it could not be identified either with lactic acid, pyruvic acid and the components of the citrate cycle, or with maleinic and citraconic acid. Oestrous secreta from eighty-four living dairy cows and sixty slaughtered animals were used as test material. The material from living cows was collected directly from the os of the cervix by aspiration with a syringe provided with a catheter. Uterine mucus, oviduct fluid, and follicular fluid was collected from slaughtered animals directly from the organ. The method of paper chromatography described by Schreier & Hack (1956) was used. The absorption spectrum of the stained compound, eluted with ethanol, was measured with a Beckman spectrophotometer. The unknown compound is supposed to be closely related to or even identical with hippuric acid, which is found in exceedingly large quantities in herbivora, as a final product of the detoxication of benzoic acid and its derivates. This conclusion is based on the following considerations. (a) The comparison made between the chromatographic data of Gaffney, Schreier, DiFerrante & Altman (1954) and our own results (see Table 1). (b) The absorption maximum of the substance tested by Gaffney et al. (1954) was at 460 m\g=m\,while that of the substance deriving from the secreta and the absorption maximum of hippuric acid under similar conditions were found at 475 m\g=m\(see Gaffney et al. Fig. 1 and Text-figs. 1 and 2). (c) In-vivo load tests also prove that the investigated substance is a benzoic acid derivative. Sixty to 90 mg/kg of sodium benzoate was administered intravenously to five cows. The substance tentatively identified as

A heparan sulphate sulphotransferase was partially purified from an ox lung homogenate by (NH(4))(2)SO(4) precipitation. Various glycosaminoglycans were assayed as sulphate acceptors with this enzyme. The highest acceptor activity was obtained with desulphated heparin and heparan sulphate, which indicates that sulphate transfer may be to free amino groups of the substrate. Some heparan sulphate was (35)S-labelled by incubation with the enzyme and re-isolated. On treatment of this heparan [(35)S]sulphate with nitrous acid and separation of the degradation products on Sephadex G-15, a major peak of radioactivity was obtained, and identified as [(35)S]sulphate by high-voltage electrophoresis at pH5.3. The [(35)S]sulphate is believed to be derived from N-[(35)S]sulphated groups of heparan [(35)S]-sulphate. That the ox lung preparation contained an N-sulphotransferase was confirmed by the isolation of 2-deoxy-2-[(35)S]sulphoamino-d-glucose as the major product from the flavobacterial degradation of heparan [(35)S]sulphate.

1. When DNA is hydrolysed with formic acid for 30min. at 175 degrees and the hydrolysate is chromatographed on paper with propan-2-ol-2n-hydrochloric acid, in addition to expected ultraviolet-absorbing spots corresponding to guanine, adenine, cytosine and thymine, an ultraviolet-absorbing region with R(F) similar to that of uracil can be detected. Uracil was separated from this region and identified by its spectra in acid and alkali, and by its R(F) in several solvent systems. 2. Cytosine, deoxyribocytidine and deoxyribocytidylic acid similarly treated with formic acid all yielded uracil, as did a mixture of deoxyribonucleotides. 3. Approx. 4% of deoxyribonucleotide cytosine was converted into uracil by the formic acid treatment.

Protein separation patterns of milk produced by cows fed normal or high energy diets during lactation were studied. Fractionation methods were column chromatography on DEAE-cellulose, paper electrophoresis in collidine acetate buffer at pH 6.8, and polyacrylamide gel electrophoresis at pH 8.3. Each cow has a characteristic milk protein pattern which changes during lactation. Feeding produced no discermible differences in milk protein pattern.

The subunit structure of tryptophanyl-tRNA synthetase from beef pancreas (mol wt 108,000) has been studied by high speed and low speed equilibrium sedimentation, light scattering, gel electrophoresis, and gel filtration. All of these methods give a molecular weight of 58,000 ± 5,000 for the protein after denaturation in 6 m guanidine hydrochloride or in sodium dodecyl sulfate. Gel electrophoresis at different pH levels, after denaturation of the enzyme by 8 m urea or after dissociation by alkylation or oxidation of all of the —SH groups of the protein, shows a single polypeptide component; these data indicate that the subunits have the same molecular weight and the same charge. Hydrolysis of the sodium dodecyl sulfate-denatured protein by carboxypeptidases A and B indicates that the COOH-terminal sequence should be -Leu-Phe-Gln; these amino acids are liberated in a ratio close to 2 eq per molecule of native enzyme. By a tritium labeling method only one type of COOH-terminal residue is detected. No free NH2-terminal amino group can be detected. Gel electrophoresis and NH2-terminal group analysis of the cyanogen bromide digestion products and fingerprints of the tryptic peptides of the enzyme are in agreement with a chemical identity of the polypeptide chains obtained after denaturation. Since it has been shown that tryptophanyl-tRNA synthetase can bind 2 molecules of substrate (tryptophan or tryptophanyl adenylate), and since no polypeptide chain of molecular weight smaller than half of that of the native enzyme could be obtained in any denaturing condition, it is concluded that this amino acid activating enzyme in its native state is a dimer made up of 2 identical subunits.

A short glycopeptide was isolated from bovine χ-casein by enzymic digestions, filtrations on Sephadex G-25 and was electrophoretically homogeneous. An O-glycosidic linkage was characterized between a residue of threonine and N-acetylgalactosamine. By chemical and enzymic proce- dures, the following formula was established: where NeuNAc =N-acetylneuraminic acid. The phosphoserine residue of χ-caseinoglycopeptide was characterized in the short glycopeptide which was localized in the C-terminal part of χ-casein. Thr-X-Pro might be suggested as “code sequence” for glycopeptides containing an O-glycosidic linkage.

Degradation of 17, 21-dihydroxy-20-ketone side-chain of cortisol by lens homo-genate was investigated and a fairly active degradation of adrenocortical steroid in the lens was verified. The assay system required the addition of nicotinamide adenine dinucleotide. Optimal pH, optimal temperature and the optimal medium composition for activity were also determined. The degradation product of corti-sol after loss of the side-chain was identified to be 11β-hydroxyandrost-4-ene-3, 17-dione by paper chromatography and scanning method using cortisol-4-14C as substrate.

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.

This work was conducted to further develop methodology for the chemical assay of estrogen in bovine urine. The method includes use of 14 C internal standards, a combination of enzyme and acid hydrolysis, paper chromatography, and fluorimetry for the assay of four estrogens in bovine pregnancy urine. Enzyme hydrolysis liberated approximately 90% of the 17 α-estradiol but only about 10% of the estrone as compared with enzyme followed by acid hydrolysis. Approximately equal amounts of 17 β -estradiol and an estriol-like compound were liberated by enzyme and acid hydrolysis. Three of the compounds, 17 α-estradiol, 17 β-estradiol and estrone, were positively identified by paper chromatographic mobilities, sulfuric acid and absorption spectra, and biological potencies. Additional proof of structure for 17 α-estradiol was provided by the Kagi-Miescher and Haenni-Iron-Kober tests. Chromatographic studies suggest the presence of 16,17-epiestriol, but inadequate quantities of the hormone prevented identification.

Milk-protein fractionation methods were studied with anion exchange chromatography, paper, and gel electrophoresis and optimal separation conditions were established. The best results were obtained when chromatographic separations were on DEAE-cellulose column (35 × 2.5 cm) with either 5 to 6 ml of skimmilk or 20 ml of whey applied and gradient eluted; paper electrophoretic analyses of either 60 µliters skimmilk or 120 µliters whey on Whatman 3 mm paper strips were performed at pH 6.8 in .067 M collidine acetate buffer and at a potential of 10.6 v/cm for 16 hours at 4 C; and polyacrylamide gel electrophoretic analyses of .3 mg protein per tube were performed at pH 8.3 and at 3.75 mamp per tube of 7.0 × .5 cm for 50 minutes at 21 to 25 C. Skimmilk protein fractions were identified with casein and whey protein markers. Attention is drawn to the effects of cold preservation of the samples at 4 C, −17 C and −78 C, and of freeze-drying on the milk protein patterns.

In order to solve a problem that there is the different predilection of atherosclerosis depending on location of artery and/or race, the studies on energy-metabolism of the bovine artery were undertaken.In a previous paper, it was reported that oxygen uptake, the incor-poration of Pi32 into the adenine nucleotides, the content of DNA, and the nucleotide content of the sliced coronary artery of cattle were almost twice of those of aorta.In this paper, column chromatographic analyses of the nucleotides and nucleosides of the sliced coronary artery and aorta of cattle were studied and compared with those of bovine myocardium and rabbit skeletal muscle. And the effects of incubation for 60min. at 37°C were also investigated.The contents of nucleotides and nucleosides of the coronary artery were 1.5 and 0.2 folds of those in aorta and myocardium respectively, and approximately 90% of nucleotides were adenine nucleotides. During the incubation, approximately 40% of nucleotides and nucleosides were released into the reaction medium. Approximately 60 to 70% of the released nucleotides and nucleosides were Hypoxanthine in the coronary artery.