Specific Color Reaction Research Articles

Pectic enzymes associated with the softening of tomato fruit

Some degree of softening of maturing tomato fruit takes place before the usually accepted mechanism for the loss of firmness, involving the sequential action of pectin esterase and polygalacturonase, is operative to any extent. The possibility that pectin and pectic acid transeliminases were implicated, providing an alternative pathway for the breakdown of pectin in the tomato, was investigated. Direct spectrophotometric determination of these enzymes proved unreliable and the thiobarbituric acid test, which should give a specific colour reaction with the products, was negative. It is concluded that transeliminases do not play a significant part in the softening of tomato fruit either prior to or during ripening.

Monogalactosyl diglyceride and digalactosyl diglyceride from commercial rapessed “lecithin”

AbstractBy silicic acid column separation small quantities of glycolipid‐rich fractions have been isolated from commercial Swedish rapeseed “lecithin.” Major components in these fractions are monogalactosyl diglyceride and digalactosyl diglyceride which have been identified by Rf on thin layer chromatography specific color reactions and IR spectrometry. The fatty acid patterns of the galactolipids are different from those of the major phospholipids of the same product, with less of palmitic and oleic and considerably more of linolenic acid.

A sensitive and specific colour reaction for the detection of free indole in plant material

A sensitive and specific colour reaction for the detection of free indole in plant material

A calorimetric method for the determination of 2-phenylphenol residues in citrus fruits.

A colorimetric method for the determination of 2-phenylphenol residues in citrus fruit, based on a specific colour reaction given by 2-phenylphenol with sulphuric acid, trace amounts of formaldehyde and iron(III), is described. 2-Phenylphenol is extracted from the fruit with chloroform, transferred into an alkaline medium, and, after acidification of the latter, re-extracted with chloroform. The extract is purified with 86·3 per cent. w/w sulphuric acid and the 2-phenylphenol is then determined colorimetrically. The method permits the determination of small amounts of 2-phenylphenol of the order of 0·05, 0·3 and 0·1 p.p.m. in the pulp, peel and whole fruit, respectively. The recovery of 2-phenylphenol varies between 95 and 106 per cent.

Purification and properties of D-gluconate dehydratase from Achromobacter.

d-Gluconate dehydratase fromAchromobacter, grown ond-gluconate, was purified 100-fold by a procedure involving ammonium sulfate fractionation and preparative acrylamide gel electrophoresis. The purified enzyme appeared to be homogeneous by disc gel electrophoresis. It is an inducible enzyme with an optimal activity in the pH region 8.4–8.8, a Km value of 2.08 × 10−2 m ford-gluconate and a molecular weight of 270,000 ± 25,000. Only C5 and C6 aldonic acids possessing al-threo configuration at C2 and C3 are dehydrated. The dehydration products ofd-gluconate,d-xylonate,d-galactonate,d-fuconate andl-arabonate were identified as 2-keto-3-deoxy compounds by specific colour reactions and thin layer chromatography. Onemm Mg+ + is a powerful activator, 0.1 mm Mn+ + activates poorly and EDTA inhibits. Glutatione, dithiothreitol and mercaptoethanol had no effect, althoughp-chloromercuribenzoate (0.01 mm) decreased enzyme activity.

A new specific colour reaction for substituted aromatic aldehydes with diphenylamine and its use for their detection and determination

A new highly selective colour reaction for the detection and determination of substituted aromatic aldehydes with diphenylamine in presence of hydrochloric acid is described. Successful colorimetric determinations of milligram amounts of anisaldehyde, salicylaldehyde, p-dimethylaminobenzaldehyde and o-vanillin were achieved. Substituted aromatic aldehydes containing methoxy or hydroxy groups form a red colour and the others containing a nitrogen atom form green coloured products on heating with diphenylamine and concentrated hydrochloric acid. Tentative mechanisms for the two colour reactions are proposed.

Routine identification of drugs of abuse in human urine : II. Development and application of the XAD-2 resin column method

The methods described in this report were developed and applied to the detection of drugs of abuse in human urine in a laboratory analyzing in excess of one thousand urines per day. Routinely 2 ml of urine were analyzed fluorimetrically for morphine and quinine. The positive morphine and/or quinine samples were confirmed by subjecting 15-ml aliquots of urine to acid hydrolysis, extracting and applying the extract to thin-layer chromatograms followed by specific chromogenic spray reagents. 25 ml of urine were applied to polypropylene columns containing 1.1 g of Amberlite XAD-2 resin. The flow rates were controlled with the hydraulic flow control apparatus and the resin eluted with 15 ml chloroform—isopropanol (3:1). The organic solvent extracts were shaken with 1 ml of saturated sodium bicarbonate and the separated organic phases evaporated to dryness in a water bath under a stream of air. The residues were dissolved in methanol and applied equally to silica gel plates and sheets. The plates were developed in ethyl acetate—methanol—water—ammonia (85:10:3:1) and the sheets in chloroform—methanol—ammonia (90:10:1). The plates were sprayed with 0.3% ninhydrin—acid, 5% H 2SO 4, iodoplatinate and p-nitroaniline followed by NaOH. The Polygram sheets were sprayed with 0.4% ninhydrin—acetone, HgSO 4, diphenylcarbazone, NaI and iodoplatinate. The opiates, opioids, barbiturates, amphetamines, phenothiazines, antihistamines and minor tranquilizers were detected by specific color reactions and R F values. The plates and sheets were cross-compared for final interpretation of the results. These methods were unusually simple, extremely rapid, inexpensive and very effective in detecting psychoactive drugs at concentrations ranging from 0.1 to 3 μg/ml of urine.

Paraquat poisoning.

This paper deals with the development of methods for the detection and determination of the weed‐killer, paraquat, in body fluids (urine, blood, gastric aspirate, dialysates) and tissues. These have been obtained from 6 cases of paraquat poisoning (2 of which were fatal) admitted to hospital and from several other negative cases. A specific colour reaction is described for the detection and determination of paraquat. Thus when sodium hydrogen carbonate followed by sodium dithionite is added to the test solution, a blue colour is produced. The detection limit is about 1 microgram/ml. Following admission to hospital shortly after the ingestion of paraquat, the reaction can be applied directly to diluted urine and diluted (filtered) gastric aspirate. Paraquat can be detected in small quantities in urine for quite a long period after ingestion. It is necessary, however, to use a column of a cation exchange resin to achieve concentration and also to remove any interfering substances. Under these conditions, it is preferable to assess the absorbance of the blue colour on a recording spectrophotometer. The concentration of paraquat in the blood serum is extremely low and only detectable shortly after ingestion. It has been extremely difficult to detect any traces of paraquat in dialysates (haemo‐ and peritoneal). Depending on the interval of time between ingestion and death, paraquat may be detected in some tissues e. g. liver and kidney. Using the cation exchange resin technique, paraquat in quantities of 50 to 100 micrograms added to 100 ml of urine can be recovered with an accuracy of 86 to 91 %.

Studies on the histidine residues of rabbit muscle myokinase

1. 1. Myokinase (ATP-AMP-phosphotransferase, EC 2.7.4.3) contains no methylated histidine residues. 2. 2. The reagent 5-diazonium-1-H-tetrazole reacted preferentially with the thiol groups of myokinase. When the SH groups were protected against azotation, the tyrosine and lysine groups reacted before histidine. As far as specific color reactions are concerned, like in other proteins ideal “bisazohistidine” plateaus were found but they were largely due to the absorption of monoazotyrosine. 3. 3. Myokinase could be reversibly inactivated by monodansylation. In monodansylmyokinase one histidine residue was protected against photochemical oxidation and against azotation. This result must be interpreted with care because dansylation leads to dimerization of myokinase. 4. 4. From photochemical oxidation experiments it follows that in myokinase there is no correlation between the loss of activity and the oxidation of the “fast” histidine. Maximally one of two “slow” histidines can be essential for enzyme activity. 5. 5. The results of photochemical oxidation confirm that two methionine residues may be involved in substrate binding and/or catalysis. 6. 6. A “destruction-reduction method” for the determination of methionine sulfoxide in proteins is introduced. It is based on the selective destruction of methionine by CNBr and the reduction of methionine sulfoxide by mercaptans during the acid hydrolysis of the protein.

Formation of 2-amino-2-deoxy- d-ribose 5-phosphate and 2-amino-2-deoxy- d-lyxose 5-phosphate by extracts of Streptomyces alboniger

Dialyzed supernatant from sonic-treated cells of a puromycin-producing strain of Streptomyces alboniger has been shown to catalyze the synthesis of two new amino pentose phosphates from d-ribose 5-phosphate and NH 4+ at pH 7.5. The amino pentose phosphates, after isolation and separation by ion-exchange chromatography, were identified as 2-amino-2-deoxy- d-ribose 5-phosphate and 2-amino-2-deoxy- d-lyxose 5-phosphate on the basis of the following criteria: (a) formation of glycolaldehyde phosphate upon periodate oxidation; (b) chromatographic behavior in different ion-exchange resin systems and on paper after enzymatic dephosphorylation; (c) isolation of erythrose and threose, respectively, after ninhydrin degradation; (d) specific color reactions before and after dephosphorylation; and (e) infrared spectroscopy. No evidence was found for the occurrence of 2-aminopentoses in acid hydrolysates of S. alboniger.

Investigations on the Chemistry of Heparin: VI. Position of the sulfate ester groups

Abstract The position of the O-sulfates in heparin was investigated by methylation procedures. Heparin was converted to the acetamido derivative and permethylated with dimethyl sulfate in aqueous alkali. Other methylation methods in organic solvents were also used. The carboxyl groups in the uronic acid moieties were then reduced and the resulting permethylated polysaccharide was hydrolyzed. The products were identified either as crystalline derivatives or by chromatography and specific color reactions. These were characterized as 3-O-methyl-d-glucose, 2,3-di-O-methyl-d-glucose, 3-O-methyl-d-glucosamine, glucosamine, and 3,6-di-O-methylglucosamine. On the basis of the amounts isolated, it is concluded that about one-third of the glucuronic acid moieties in heparin are sulfated on carbon atom 2 whereas the rest are not sulfated. Most of the glucosamine in heparin contains sulfate ester groups on carbon 6. Glucosamine units which are not sulfated and 3,6-di-O-sulfoglucosamine units are also present in small proportions. It is proposed that the degree of sulfation of the individual units in the heparin polymer is not uniform and that it may vary in different parts of the chain.

Deposition of dietary epoxides in tissues of rats.

AbstractEpididymal fat pad lipids from rats fed trivernolin at the 4.8% level in the diet for 90 days were found to contain 6.1% epoxyoleic acid. No epoxides were detected in the serum lipids and only trace amounts were found in the liver lipids. TLC, GLC and a specific color reaction with picric acid were used to identify this fatty epoxide in the tissues. Epoxyoleic acid was shown to be present as a mixture of predominately monovernoloyl triglycerides with some divernoloyl triglycerides and small amounts of trivernolin. Lipase hydrolysis of the first two triglycerides has demonstrated that the vernoloyl groups are present mostly in the 1,3 positions. In a separate experiment, rats were fed cholesterol epoxide at the 0.5% and 1.5% levels in the diet for 90 days. TLC and GLC examination of lipids from these rats failed to reveal the presence of any cholesterol epoxide. Only one‐half of the sterol fed could be accounted for in the fecal lipids.

Application of specialized techniques for specific staining of peptide maps on various media

Combinations of specific color reactions useful in peptide mapping on W h a t m a n filter papers have been described in a previous communicat ion 1. This paper introduces a new combinat ion of specific reactions for staining peptide maps on W h a t m a n No. 3MM paper 2, and to a limited extent, on thin-layer plates. Cellulose plates used were E. Merck thin-layer chromatographic cellulose-precoated plates. Silica gel plates were prepared ~ from Merck silica gel G and Koch-Light amylopectin. Staining ofpeptide maps on paper. An optimal combination of several staining reactions on one peptide map is desirable, in order to eliminate variations seen in duplicates and to conserve time and materials. The new staining sequence described here combines 5 reactions on a single 3MM paper. Best results m a y be obtained on a peptide map prepared from an aliquot of digest representing at least o . I / ,mole original protein. A distinct advantage of this staining sequence is the combination of a highly sensitive test for arginine with the Pauly reaction for histidine and tyrosine; the Sakaguchi reagent for arginine and the Pauly reagent are incompatible on the same paper 1. A disadvantage is tha t the tyrosine reaction is weak as applied here ; occasionally it does not work at all. These reactions are performed in the order in which they are described. To avoid confusion, the spots are marked after each reaction, and the results incorporated on a composite tracing. I. The platinic iodide reaction for reducing sulfur is performed as described previously 1. The paper must be thoroughly dried after electrophoresis, since remaining traces of the solvents used, as well as pyridine or collidine in the atmosphere, seem to interfere with this reaction.

Identification of polyhydric alcohols on thin-layer chromatograms with 2-thiobarbituric acid

A procedure is outlined for identification of polyhydric alcohols by thin-layer chromatography. Those with similar RF values are identified by their specific colour reactions with 2-thiobarbituric acid.

A specific color reaction of tyrosine and tryptophan

Tyrosine and tryptophan were spotted on thin layer of Japanese acid clay and the plate was air-dried at room temperature.Ninhydrin solution(0.5% ninhydrin in 95% ethanol) was sprayed on that plate, which then was heated at 100110°C for 10 min.After cooling, a blue or dark green stain appeared within 12 hours.The blue stain of tyrosine turned red by a drop of 3N NH4OH, and this turned blue by a drop of 3N HCl. On the other hand, the color of tryptophan did not changed by NH3 and hence by HCl. The tests were not interfered from other amino acids.

Analytical applications of ternary complexes—VI : Spectrophotometric determination of trace amounts of palladium

Extremely sensitive and specific colour reactions in both aqueous and organic solutions are described for the spectrophotometric determination of palladium(II) ions. The methods are based upon ternary complex formation with 1,10-phenanthroline or pyridine, and Rose Bengal Extra. Themolar absorptivities are 5 × 10 4 (aqueous) and 1.25 × 10 5 (organic) in the presence of a 1000-fold molar excess of EDTA as mass masking agent. Under these conditions there is negligible interference with the pyridine-Rose Bengal Extra system from the 22 cations (including silver, gold and platinum) and seven anions investigated. Both colour reactions take place within 15 min and the complexes are stable for long periods.

ON THE QUESTION OF OCCURRENCE OF INDOLE‐3‐ACETIC ACID IN PINUS SILVESTRIS

The inner bark of Pinus silvestris L. contains a natural acidic growth promoter which stimulates elongation of wheat coleoptile sections and oat mesocotyl sections, produces oat coleoptile curvature, and is not effective in an oat first‐leaf increment test. It occupies the same or a closely related RF to that of authentic IAA if rechromatographed with seven partition solvents. Pine auxin described by Fransson could not be detected by spraying with reagents known for developing the color reaction of IAA. Natural pine resin from wounds, when added to test solutions, affects several characteristic features of authentic IAA which are commonly used for its identification. These are: (a) position on chromatograms (RF), especially if 70% ethanol is used as the partition solvent, (b) amount of growth promotion in bioassays, and (c) ability to develop specific color reactions with IAA on chromatograms with known reagents. Interaction with pine resin or other related substances which could not be separated from the extract by conventional partition methods may be responsible in previous studies for the difficulties in identifying the auxin of Pinus silvestris as an indole‐3‐acetic acid.

Structural Comparison of Polymorphic Hemoglobins of Deer with Those of Sheep and Other Species

Abstract The structure of deer hemoglobin III, associated with sickling of the erythrocytes of Florida white tailed deer, is compared with that of deer hemoglobin V, the presence of which precludes sickling of erythrocytes under appropriate conditions, by peptide mapping and amino acid analysis techniques. Peptide mapping techniques, including combined specific color reactions, have been utilized to relate hemoglobin peptides in the deer hemoglobins to those of several species for which the amino acid sequence is known. Certain structural regions of the α and β polypeptide chains of deer hemoglobin are closely homologous to the corresponding regions in the α and β chains of these other species, as illustrated by a graph based upon the peptide mapping studies and known sequences. The tryptic peptide mapping pattern of the α chains of deer hemoglobins III and V is nearly identical with that of the sheep α chains from the three polymorphic sheep hemoglobins. In contrast to the similarity of the α chains of these two species, the β chains exhibited numerous inter- and intraspecies differences. However, the marked variations of the β chains among the polymorphic hemoglobins of the ruminants are largely confined to distinct regions of these β chains. Although the significance of a linear comparison of the homologous regions is not apparent, the relationship of these regions is clarified by use of a three-dimensional model.

3 The Pharmacology of Heparin and Heparinoids

This chapter explores that heparin and heparinoids constitute a group of compounds that have many remarkable pharmacological properties. The chapter demonstrates that heparin binds or complexes with proteins and other bases and so modifies their biological activity. As a result, heparin is able to release or activate enzymes such as the lipoprotein lipase, to inhibit hormones such as cortisone and aldosterone, to detoxify toxic agents, and to bind histamine in body cells. It was also discovered that heparin is a highly sulphated polysaccharide and that it gives a specific color reaction with dyes—the metachromatic reaction. This resulted in (1) the association of heparin with the naturally occurring mucopolysaccharides; (2) the preparation of other sulphated polysaccharides, heparinoids; and (3) the identification of the mast cells as heparinocytes. The chapter reviews that heparin is a term that may be used in three ways. It is used for any heat-stable naturally occurring anticoagulant activity; for a commercial product of varying composition with anticoagulant and other activities; for a distinctive group of sulphated mucopolysaccharides, which can be distinguished by certain precise chemical and biological properties-formation of a crystalline barium salt with well defined chemical composition; low viscosity in aqueous solution; exceptional ability to combine with proteins to alter their physical and biological properties and produce metachromatic colours with basic dyes. Heparinoids are sulphated polysaccharides, prepared semi-synthetically by sulphation of partially degraded polysaccharides or occurring naturally in plant or animal tissue. They possess the properties described for heparin mucopolysaccharide.

The use of diphenylamine-aniline-phosphoric acid reagent in the detection and differentiation of monosaccharides and their derivatives on paper chromatograms

The diphenylamine-aniline-phosphoric acid reagent, as modified by V ítek, has been used for the detection of various monosaccharides and their derivatives. Under the defined conditions, the reagent gives specific color reactions and can be used (1) to distinguish between sugars of different structure and (2) to detect various non-reducing sugar derivatives with acid-labile blocking groups.