Tris-hydrochloric Acid Buffer Research Articles

Purification and kinetic study of bone and liver alkaline phosphatase isoenzymes in the dog

To study bone and liver alkaline phosphatases (ALP) in the dog, tissue samples were obtained immediately after death from dogs apparently involved in road traffic accidents. Tissue samples were homogenized, and extracted protein was subjected to diethylaminoethyl cellulose–Sephadex column chromatography equilibrated previously with 0.01 M Tris-hydrochloric acid buffer (pH 7.2) and eluted with linear gradient of ionic strength from 0 to 0.4 M NaCl. Fractions with peak activity of ALP were considered a source of purified enzyme. ALP activity was measured by a kinetic method using p-nitrophenylphosphate as a substrate. The kinetic study was performed using a constant concentration of purified isoenzymes and different concentrations of the substrate. Precise Km values for liver and bone ALP were identified using direct linear plot (Eisenthal–Cornish-Bowden plot). Our study showed that the Km value of liver isoenzyme was 13 times greater than that of bone isoenzyme (3.3 mmol and 0.25 mmol, respectively). This may be due to the physiological role of bone ALP in preparing phosphate for bone mineralization and the retention of phosphates in mineral complexes with calcium in bone tissues.

Determination of sperm-antibodies in sera of sterile women by affinity-chromatography (affinity-chromatography of sperm-antibodies).

The total amount of humoral sperm antibody protein is determined following its isolation by affinity-chromatography on spermatozoa. Serum proteins are eluted by Sorensen phosphate buffer. Antibodies attached to spermatozoa suspended in Sephadex G 75 columns resist elution by Sorensen buffer. They can be quantitatively washed out by 8 ml Tris HCl-buffer pH 8,9 and be determined as proteins by the Folin-Ciocalteu method.

Chemical detections of active compounds in Melilotus indica extracts

The crude extracts of the leaves of melilotus indica which collected in two dates (midFebruary, mid March) have been prepared by three solvents (Distilled water, ethanol 80% and chloroform) and three buffer solutions (Sodium acetate buffer, Sodium phosphate buffer and Tris-hydrochloric acid buffer), the greater percentage of crude extracts achieved by phosphate buffer which were 47.3% and 29.2% for second and first dates, respectively, while the lowest percentage of crude extracts achieved by chloroform extracts which were 13.2% and 10.0% for second and first dates, respectively. The qualitative chemical detections for active compounds in crude extracts revealed a positive results for the saponins, tannins, coumarins, flavones and glycosides, while the detections revealed a negative results for the alkaloids, resins and volatile oils. The active compounds in crude extracts prepared by solvents and buffer solutions were studied by thin layer chromatography (TLC), the solvent extracts were contains five compounds with relative flow (RF) 0.65, 0.4 for coumarin and umbelliferon, respectively, and 0.5, 0.27, 0.2 which belong to simple coumarins compounds in melilotus indica, while buffer solution extracts were contains two compounds with RF value 0.65 and 0.4 for coumarin and umbelliferon, respectively.

Hydrolysis of Soy Phosphatidylcholine with Phospholipase A2

Hydrolysis of soy phosphatidylcholine (S-PC) to lysophosphatidylcholine by using phospholipase A2 was conducted under various conditions without stirring. The degree of hydrolysis was determined based on the amounts of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) by high performance liquid chromatography (HPLC). HPLC was simple, rapid and reproducible for quantitative analysis of PC and LPC in S-PC hydrolysate.The hydrolysis of S-PC depended on reaction temperature, reaction time, CaCl2 concentration, and buffer.S-PC was more easily hydrolyzed by Tris-hydrochloric acid than by H3BO3-KCl-NaOH, NaH2PO4-Na2HPO4 or Na2HPO4-citric acid as buffer.Optimum S-PC hydrolysis conditions were as follows : Tris-hydrochloric acid buffer; pH 8, CaCl2; 0.3 wt%/S-PC, 60°C, S-PC/aqueous medium; 1/2 (g/mL), phospholipase A2; 10IU.

Micro Enzyme-Sensor with Osmium Complex and a Porous Carbon for Measuring Uric Acid

Abstract A micro enzyme-sensor using uricase (EC 1.7.3.3) was developed for measuring uric acid. For this, a sheet of porous composite was made from acetylene black and Teflon emulsion and was soaked in Zonyl FSN fluoro-carbon surfactant and Nafion. The 50 μ m platinum micro-electrode was etched in hot aqua regia to create a cavity at its tip and this cavity was packed with porous composite material by pushing the tip into small lumps of the material. The redox mediator [Os(bpy)3 (PF6)2] was monitored by cyclic voltammetry. The porous micro-sensor was dipped overnight in Tris-hydrochloric acid buffer of pH 8.0 containing enzyme for adsorption of uricase at 4°C. The effects of pH, and temperature on measurements of uric acid with the sensor were examined, the calibration curve for measurement of uric acid under optimum conditions was determined and the effect of concomitant compounds were investigated. Under optimum conditions, the response of the micro-sensor was linear in the concentration range of 0.101 - 1.93mM uric acid. The uric acid concentrations in commercial preparations and human serum were also determined with this sensor.

Determination of free and total catecholamines in human urine by HPLC with fluorescence detection.

A simple and highly sensitive method for the determination of free and total (free + conjugated) catecholamines (norepinephrine, epinephrine and dopamine) in human urine is described which employs HPLC with fluorescence detection. Conjugated catecholamines (sulfate form) are hydrolyzed by a sulfatase-mediated reaction to the corresponding free amines. After cation exchange chromatography on a Toyopak IC-SP S cartridge, catecholamines and isoproterenol (internal standard) in urine samples were converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. These compounds were separated within 8 min on a reversed phase column with isocratic elution using a mixture of water, methanol and acetonitrile containing a Tris-hydrochloric acid buffer (pH 7.0). The detection limit for each catecholamine is ca 2 fmol per 100 microL injection volume.

Cadmium-Binding Capacity of a Soybean Protein Fraction

The cadmium-binding capacity of the soybean protein fraction was determined by equilibrium dialysis. The aqueous supernatant was extracted from the soybeans with 0.01M Tris-hydrochloric acid buffer (pH 7.4) by ultracentrifugation. The protein fraction (F-I, >10, 000 in molecular weight) was separated from the extract by Sephadex G-50 chromatography. Binding measurement was carried out in 0.01M Tris-hydrochloric acid buffer (pH 7.4). The binding data were analyzed according to the Scatchard equation. A Scatchard plot of the binding data showed several phases, indicating heterogeneity of the binding sites. The maximum amount of cadmium bound by F-I was calculated to be 43, 000μg/g. When cadmium was added to the aqueous supernatant, five cadmium-binding fractions (at molecular weights of>100, 000, 75, 000, 38, 000, and two low-molecular species) were detected by chromatography on Sephadex G-100. The major cadmium-binding fraction in F-I was the macromolecular species of>100, 000 in molecular weight.

High-performance liquid chromatography of plasma catecholamines using 1,2-diphenylethylenediamine as precolumn fluorescence derivatization reagent

A simple, rapid and highly sensitive method for the determination of catecholamines (norepinephrine, epinephrine and dopamine) in human plasma is described which employs high-performance liquid chromatography with fluorescence detection. After cation-exchange chromatography on a Toyopak SP cartridge, the catecholamines and isoproterenol (internal standard) in 500 μl of plasma are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine in aqueous acetonitrile. These compounds are separated within 8 min on a reversed-phase column, TSK-gel ODS-120T, with isocratic elution using a mixture of water, methanol and acetonitrile containing a Tris—hydrochloric acid buffer (pH 7.0). The detection limit for each catecholamine is ca. 2 fmol in a 100-μl injection volume. N-Methyldopamine can also be used as internal standard.

A Separative Determination of Methylguanidine and Agmatine in Foods by High Performance Liquid Chromatography with Fluorescence Detection

A sensitive and simple separation method for methylguanidine (MG) and agmatine (AGM) in various foods by high performance liquid chromatography with a fluorescence detector is described. Food samples were homogenized in 4% trichloroacetic acid solution and filtered. The guanidino compounds were extracted with n-butanol from the filtrate under alkaline conditions and re-extracted with 1N hydrochloric acid. They were converted into fluorescent derivatives by reaction with benzoin under alkaline conditions. Guanidino compounds were separated and determined on a Nucleosil 5C18 column with methanol -0.5M Tris-Hydrochloric acid Buffer (pH 8.6) (8:2) as the mobile phase. The recoveries of MG and AGM added to smoked dried fish and wiener sausage were 85.0% (MG), 82.5% (AGM) and 88.2% (MG), 82.3% (AGM), respectively. The limits of determination were 0.1ppm for MG and 0.5ppm for AGM. Smoked-dried bonito (Katsuo-bushi) contained 12 to 238ppm of MG. The amounts of MG and AGM determined in almost all samples were small or below the detection limits.

High performance liquid chromatography of free catecholamines in urine using 1,2-diphenylethylenediamine as new precolumn fluorescent derivatization reagent.

A simple, rapid and highly sensitive method for the determination of free catecholamines(norepinephrine, epinephrine and dopamine) in urine is described which employs high performance liquid chromatography with fluorescence detection. Catecholamines and isoproterenol added as an internal standard in 100 μl of urine, after chromatography on a small column of CM-Sephadex C25, are converted into the corresponding fluorescent compounds by the reaction with 1, 2-diphenylethylenediamine in aqueous ethanol. These compounds are separated within 7 min on a reversed phase column, TSK-gel ODS-120T, with isocratic elution using aqueous dioxane containing a Tris hydrochloric acid buffer (pH 7.0). The detection limits of norepinephrine, epinephrine and dopamine are 20, 30 and 40 fmol in a 50 μl injection volume, respectively.

High-performance liquid chromatography of guanidino compounds using benzoin as a pre-column fluorescent derivatization reagent

A high-performance liquid chromatographic method is described for the simultaneous separation of nine guanidino compounds of biological importance. Guanidino compounds are converted into the corresponding fluorescent derivatives by reaction with benzoin, and separated within 25 min on a reversed-phase column, μBondapak Phenyl, with linear gradient elution using aqueous methanol containing a Tris—hydrochloric acid buffer (pH 8.5). The method is simple, rapid and sensitive; the lower limits of detection for the guanidino compounds are 20–100 fmol in a 100-μl injection volume.

Distribution of Zinc and Purification of Copper Complex in the Water-soluble Low-molecular-weight Fraction of Soybeans

The water-soluble fraction of soybean was extracted with 0.01M Tris-hydrochloric acid buffer (pH 7.4). The low-molecular-weight fraction (F-II, MW<10, 000) was separated from the soluble fraction by gel filtration on Sephadex G-50. The distribution of zinc (Zn) and the chemical form of copper (Cu) were studied.The concentrations of Cu and Zn in the water-soluble fraction were 26.6 and 107ppm, respectively. Zn in the water-soluble fraction was observed only as the bound form. Zn in F-II was always eluted together with Cu in F-IIB by gel filtration on both Sephadex G-25 and G-10. The binding affinity of Zn in F-IIB was found to be less than that of Cu. When Cu was added exogenously to the water-soluble fraction, Zn in F-IIB shifted to F-I. However, the addition of Zn did not cause a shift of Cu in F-IIB.The purification of the Cu complex in F-IIB was carried out by the application of anion exchange column chromatography (AG 1×2, DEAE-Sephadex A-25). Cu and Zn in F-IIB were held on the AG 1×2 column and both were eluted with 0.01M acetic acid. The dissociation of Zn from the ligands was observed in the chromatography, but Cu was always eluted together with nitrogen-containing materials which were ninhydrin-positive. The Cu complex in F-IIB gave a blue band on the DEAE-Sephadex A-25 column, but after purification, it did not have any absorption spectrum in the visible region. The Cu complex had the same elution point on Sephadex G-10 before and after the purification.The nitrogen/Cu ratio in the partially purified Cu complex was approximately 4-6 mole/mole. The UV spectrum of the partially purified Cu complex showed strong bands at 250nm and a shoulder at around 225nm. Amino acid analysis gave several major peaks (Asp, Glu, Thr, Ser, Gly, Cys and 1-MeHis). The partially purified Cu complex showed a characteristic ESR spectrum of Cu (II) complexes.

Chemical characterization of the hen eggshell matrix: isolation of an alkali-resistant peptide

The eggshell matrix was obtained from hen eggshell using EDTA solutions. The water-soluble organic material was separated on a DEAE-cellulose column equilibrated with 8 M urea using Tris-hydrochloric acid buffer as cluent with a linear gradient of sodium chloride. Five main fractions were obtained which differ in amino acid composition and sugar contents. As is shown from the uronic acid content, the first two fractions eluted from the column are glycoproteins, while other three contain proteins and glycosaminoglycans. From the alkaline hydrolysate of the eggshell matrix, a peptide was isolated which is composed of aspartic acid, threonine, serine, glutamic acid, proline, glycine and alanine in a molar ratio of 2:1:3:7:1:3:1 with a minimum molecular weight of 2158 daltons. The calcium ion binding to this peptide was studied, at different pH values, with both free and blocked carboxyl groups, using murexide as an indicator of free Ca 2+. The importance of this acidic peptide in the calcification process of the eggshell matrix is discussed.

Distribution of and Binding Capacity for Copper in the Water-soluble High Molecular Weight Fraction of Soybeans

The water-soluble fraction of soybeans was extracted with 0.01M Tris-hydrochloric acid buffer (pH 7.4). The high molecular weight fraction (F-I, MW.>10, 000) was separated from the soluble fraction by gel filtration on Sephadex G-50. The chemical forms of copper (Cu) in F-I were investigated.F-I was separated into five components by chromatography on a Sephadex G-100 column (2.0×70cm). Most of the endogeneous copper in F-I was eluted in the highest molecular weight fraction (F-IA, MW.>100, 000), the major component in F-I. When copper was added exogeneously to F-I, further copper could bind with F-I and over half the bound copper was found in F-IA.Copper-binding affinities of F-I and F-IA were determined by equilibrium dialyses. The dissociation constants of F-I and F-IA for copper were calculated to be 2.3×10-5M and 2.0×10-5M, respectively, by fitting the Scatchard equation to the copper binding equilibrium curves. Maximal binding amounts of copper with F-I and F-IA were estimated to be 6, 000 and 5, 700ppm, respectively.Artificial [F-I]-Cu complex containing a high level copper was prepared by the addition of copper to F-I. The copper content of the artificial complex was almost constant (2, 700ppm) in several experiments. The stability of this complex were studied by equilibrium dialyses against EDTA-2Na, 2-mercaptoethanol (2-ME), organic acids (6 kinds), amino acids (15 kinds) or metal ions (10 kinds). The artificial complex was unstable to EDTA-2Na, histidine, Zn2+, Hg2+ or Cd2+. On the other hand, the complex was found to be stabilized by compounds having SH groups: 2-ME, cysteine and glutathione (reduced form).

Gel Filtration of Copper Binding Components in Soybeans

Copper binding components in soybeans were extracted with buffer solution and separated by gel filtration.1. 60% of Cu in soybeans was extracted with 0.01M Tris-hydrochloric acid buffer solution (pH 7.4). The protein content of the crude extract was approximately 63%.2. The crude extract was characterized by gel filtration on Sephadex G-50. It was separated into two fractions (F-I, m. w.>ca. 10, 000 and F-II, m. w.<ca. 10, 000). F-I contained 20ppm Cu and 80-90% protein, while F-II contained 24ppm Cu and ca. 1% protein.3. F-I was treated with enzymes. No change was found in the gel filtration profile of F-I on Sephadex G-50 after treatment with trypsin or α-amylase.4. The binding capacity of F-I to Cu was also determined. When Cu was added in batches to F-I, the amount of bound Cu increased with the amount added. When 80μg of Cu was added to 20mg of F-I, the concentration of Cu in F-I increased over 80 times (1, 700ppm) from the original level (20ppm).5. Cu in F-II was eluted together with nitrogen-containing material, which gave a positive ninhydrin reaction. There may be an interaction between Cu and peptides in F-II.

5-Dehydroshikimate reductase in the tea plant (Camellia sinensis L.). Properties and distribution.

1. A method for the extraction of 5-dehydroshikimate reductase (EC 1.1.1.25) from tea plant tissues in an active soluble state has been developed. It is dependent on the use, in the extraction medium, of an insoluble polyphenol adsorbent (Polyclar AT), which prevents the polyphenols present from precipitating all the proteins. 2. The enzyme has the following properties: pH optima at pH10.1 in glycine-sodium hydroxide buffer and at pH7.7 in tris-hydrochloric acid buffer; K(m) (NADP) 32mum and K(m) (shikimate) 0.43mm; and NADP-specificity. It was completely inhibited by 0.33mm-p-chloromercuribenzoate and this inhibition was completely reversed by 10mm-cysteine. Iodoacetate and arsenite inhibited the enzyme to a smaller extent. 3. The specific activity of the enzyme was higher in the parts of the actively growing shoot tips (third leaf>stem>second leaf>first leaf>bud) than in the mature leaves. However, the mature leaves had the greatest total activity. 4. The importance of these findings with respect to flavanol biosynthesis in tea plants is discussed.

Purification, properties and comparative specificities of the enzyme prolyl-transfer ribonucleic acid synthetase from Phaseolus aureus and Polygonatum multiflorum

1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus enzyme. One-half the enzymic activity was destroyed by preheating for 5min. at 62 degrees in tris-hydrochloric acid buffer, pH7.9. 8. All experimental evidence supports the hypothesis that azetidine-2-carboxylic acid and proline are activated by the same enzyme in Phaseolus preparations, whereas the analogue was inactive in all Polygonatum preparations. The possible nature of this different substrate behaviour is discussed.

FURTHER OBSERVATIONS ON THE PRODUCTION OF AMINO ACIDS BY RAT-LIVER MITOCHONDRIA AND OTHER SUBCELLULAR FRACTIONS.

1. Rat-liver mitochondria showed a decrease in amino acid production after preparation in 0.25m-sucrose containing EDTA (1mm), but an increase in water content. When EDTA was replaced by Mn(2+) (1mm) or succinate (1mm), both amino acid production and water content were lowered, whereas preparation in 0.9% potassium chloride caused an increase in both. 2. Amino acid production by rat-liver homogenates prepared in 0.9% potassium chloride or 0.25m-sucrose was similar (q(amino acid) 0.047 and 0.042 respectively aerobically). After freezing-and-thawing q(amino acid) values were approximately doubled, and approached that of a homogenate prepared in water. 3. All cations tested inhibited amino acid production by mitochondria, Hg(2+) and Zn(2+) being the most effective in tris-hydrochloric acid buffer. In phosphate buffer Mg(2+) and Mn(2+) had no effect. Of the anions tested only pyrophosphate and arsenate had any inhibitory effect at final concn. 1mm. 4. Iodosobenzoate (1mm) and p-chloromercuribenzenesulphonate (1mm) inhibited mitochondrial amino acid production by 70-80%, whereas soya-bean trypsin inhibitor, EDTA and di-isopropyl phosphorofluoridate inhibited by a maximum of 30%. Respiratory inhibitors had no effect. 5. Rat-liver homogenate and subcellular fractions each showed an individual pattern of inhibition when a series of inhibitors was tested. 6. Amino acid production by mitochondria was decreased by up to 50% in the presence of oxidizable substrate, apart from alpha-glycerophosphate and palmitate, which had no effect. CoA stimulated amino acid production in tris-hydrochloric acid but not in phosphate buffer, alpha-oxoglutarate abolishing the stimulation. 7. Cysteine and glutathione stimulated amino acid production by whole mitochondria by 30%, but only reduced glutathione stimulated production in broken mitochondria. 8. Adrenocorticotrophic hormone and growth hormone stimulated mitochondrial amino acid production by 21-24%, whereas insulin inhibited production by 25%. 9. Coupled oxidative phosphorylation increased amino acid production by up to 154% at 25 degrees and 40 degrees . The increase was abolished by 2,4-dinitrophenol. 10. Amino acid incorporation in mitochondria was accompanied by an increase in amino acid production, both being decreased by chloramphenicol. 11. Mitochondrial production of ninhydrin-positive material was increased in the presence of albumin. The biggest increase was noted for the soluble fraction of broken mitochondria. No increase was found in the presence of (14)C-labelled algal protein or denatured mitochondrial protein.

PREPARATION OF RAT GROWTH HORMONE.

WE have devised a simple method for the preparation of rat growth hormone from fresh frozen anterior pituitary glands. The hormone preparation was obtained in high yield and showed good biological activity while being essentially free of contamination by other pituitary hormones. Disk electrophoresis on polyacrylamide gels indicated the hormone to be relatively homogeneous. In a typical experiment 300 rat pituitary glands were homogenized for 1 min in 4 ml. of ice cold 0.3 M potassium chloride using a glass homogenizer with a ‘Teflon’ pestle. The homogenate was adjusted to pH 5.5 and stirred for 20 min at 5° C and afterwards centrifuged for 10 min at 14,000 r.p.m. The resultant supernatant was brought to an ethanol concentration of 30 per cent at −20° C and immediately centrifuged at 14,000 r.p.m. for 5 min. Additional growth hormone (10–12 mg) was recovered by extracting the residue in 0.3 M potassium chloride at pH 10. However, inconsistent biological activity has been obtained with the material so extracted. The ethanol supernatant was then dialysed, lyophilized and when assayed was found to contain the gonadotropic hormones. The ethanol precipitate was taken up in 1 ml. of tris-hydrochloric acid buffer, pH 8.6 (0.2 M tris, 0.05 M hydrochloric acid), and dialysed against four 500 ml. changes of this same buffer for a period of 4 h at 5° C. The dialysed solution was applied to a block of ‘Pevikon C-870’ (92 cm × 5 cm × 0.5 cm) equilibrated with tris-hydrochloric acid buffer, pH 8.6. A current of 400 V and 80 m.amp was applied for 18 h. After the completion of electrophoresis, the ‘Pevikon’ block was divided into 2-cm segments and each individual fraction was eluted with three 5-ml. portions of buffer using small sintered glass funnels and suction. ‘Pevikon’ particles, which passed through the filter, were removed by centrifugation. The equipment as well as the procedure used for zone electrophoresis was similar to that described by Muller-Eberhard1. The effluent diagram of a typical zone electrophoresis is shown in Fig. 1. Protein concentration was estimated by measuring absorption at 280 mµ The effluent portions were concentrated by partial lyophilization and combined into Fractions A, B and C after analysing individual eluates by disk electrophoresis. Each of the combined fractions was lyophilized and then desalted by gel-filtration on ‘Sephadex G-25’. Fraction A was found to contain from 20–25 mg of rat growth hormone. Fraction B (10–12 mg protein) contained some growth hormone, but was also found to be contaminated with Fraction C. This latter fraction, yielding 8–10 mg of protein, demonstrated no growth hormone activity. Zone electrophoresis of Fraction B resulted in the recovery of an additional quantity (5–7 mg) of rat growth hormone. Zone electrophoresis studies have also been carried out at pH 10 (carbonate Γ/2 = 0.1) with no improvement in resolution. It was noticed that additional electrophoretic components were formed on exposure to pH 10 as compared with the electrophoretic pattern which resulted from isolation at pH 8.6. The yield and biological profile of rat growth hormone (Fraction A] is summarized in Table 1. Of particular interest is the absence of significant prolactin activity.

A ribonucleoprotein from amphibian gastrulae.

DISAGGREGATION of amphibian embryos by agents which chelate calcium is succeeded by re-aggregation if the cells are returned to a medium containing calcium1. It is customary to consider that the removal of calcium from the environment of the cells is responsible for the loss of adhesion displayed in disaggregation. An attempt has been made to find out whether any organic material is removed from the embryos during disaggregation. Such organic material might lie on the cell surfaces or between the cells and assist in their adhesion, or it might be removed from within the cells during disaggregation. Batches of 250–260 late gastrulæ of Xenopus laevis were disaggregated in 0.001 M ethylene diamine tetraacetate at pH 8.03 in tris-hydrochloric acid buffer (0.001 M in tris) made up in calcium-free Holtfreter solution. The embryos were gently agitated on a rocker for 40 min. at 19–21° C., and at the end of this period the cells of the completely disaggregated embryos were allowed to settle. No evidence of cytolysis of the cells could be found. The supernatant was removed and centrifuged at 300–400 g for 10 min. A small number of intact cells, and a few yolk platelets probably of extracellular origin, formed the residue. The clear supernatant from this centrifugation was examined spectrophotometrically, and the absorption curve suggested the presence of nucleic acids. The preparation was frozen-dried.