Pigment Derivatives Research Articles

Liquid-chromatographic determination of bilirubin and its conjugates in rat serum and human amniotic fluid.

This newly developed and highly specific and sensitive procedure was applied to the determination of unconjugated bilirubin and its ester conjugates in rat serum and human amniotic fluid. Bilirubin conjugates in biological samples are converted to methyl esters by alkaline methanolysis, extracted into chloroform, and the unconjugated bilirubin and esterified pigment derivatives are fractionated by "high-performance" liquid chromatography. The separated pigments are measured spectrophotometrically. Bilirubin and its mono- and di-conjugates are readily quantitated, even in previously undetectable concentrations. Linearity was established from 0.07 to 121.2 mumol/L for unconjugated bilirubin, 0.07 to 34.6 mumol/L for the C-8 monoconjugate, 0.06 to 69.3 mumol/L for the C-12 monoconjugate, and 0.17 to 43.7 mumol/L for the di-conjugate fraction. The detection limit was 0.03 mumol/L for unconjugated bilirubin and for each monoconjugate, and 0.1 mumol/L for the di-conjugated pigment.

The Differentiation of Pigmentation in Flower Parts. IX. Changes in Pelargonidin Derivatives in Developing Petals of the Red-Flowered Genotype (llHHPrPr) of Impatiens balsamina

Summary With the aid of polyamide CC, the quantitative changes of 6 pelargonidin derivatives in petals of the red-flowered genotype (llHHP r P r ) of Impatiens balsamina were followed. Several minor compounds, previously reported and tentatively identified, were enriched and purified. Compounds formerly described as 3-glucosylglucosides have now been identified as 3-rhamnosylglucosides. Based on the changes in each pigment derivative during flower petal development a revised biogenetic pathway of 11 different pelargonidin derivatives in this tissue is proposed.

Studies on the Mechanism of the Voges-Proskauer Reaction The Crystal Structure of Pigment A'

The structure of Pigment A obtained in the Voges-Proskauer reaction was determined as 2-(4-methyl-2-morpholino-1 H-5-imidazolylmethylidene)-1, 2-dihydro-1-oxonaphthalene, by the X-ray analysis of Pigment A', a heavy atom derivative of Pigment A. Pigment A' crystallizes in the triclinic system, space group P-1, with unit-cell dimensions a=8.08Å, b=14.63Å, c=7.43Å, α=101.5°, β=97.2°, γ=92.1°, Z=2. The structure was solved by the heavy atom method and refined by the least-squares method to an R value of 0.065.

Studies on the Voges-Proskauer Reaction. VII. Pigments obtained from the Reaction with 2- and 4-Chloro-, and 2, 4-Dichloro-1-naphthols and Reactivity of Some Phenols and Naphthols

The structure of pigments derived from 2-chloro-(Pigment III) and 4-chloro-(Pigment I), and 2, 4-dichloro-1-naphthol (Pigment II), and the reactivity of some phenols and naphthols were investigated in the reaction mixture of diacetyl and 1, 1-pentamethyleneguanidine in alkaline medium. The structures of Pigments I and II were identical with 4-chloro derivative of Pigment A obtained from 1-naphthol as reported previously, and Pigment III was a para-isomer relative to the structure of Pigment I and II. It was found that Pigment III was also identical with 2-chloro derivative of Pigment B which could not be separated from 1-naphthol because of its instability. For the reactivity of phenols and naphthols, 1, 3-dihydroxy type such as 2-methyl- and 5-methyl resorcinol, 3, 5-dihydroxy benzoic acid, and 1, 3-dihydroxynaphthalene gave striking coloration in comparison with 1-naphthol, while 1, 2- or 1, 4-dihydroxy type, which is immediately oxidized to a quinone form, did not give positive coloration.

Some unusual anthocyanins occurring naturally or as artifacts

Artifacts appeared during chromatographic purification of anthocyanins in BAW (butanol-acetic acid-water, 4:1:5); they were shown to be produced by the combined action of acetic and hydrochloric acids during concentration of components eluted from the paper. The extent of their formation depended upon the nature of the sugar, e.g. cyanidin 3-glucoside formed an artifact more readily than cyanidin 3-galactoside. These and similar anthocyanins produced using propionic, butyric and valeric acids instead of acetic acid were assumed to be acylated with the appropriate acid. An artifact of similar type occurred also during solvent development (BAW) of strongly acid solutions of pelargonidin 3-glucoside but not of cyanidin or delphinidin 3-monosides. Two naturally occurring anthocyanins of similar properties found in Cichorium intybus leaves appeared to be acylated derivatives of the main pigment, cyanidin 3-glucoside.

The mechanism of action of some new deflocculating agents for pigments in paint media

Soluble basic pigment derivatives are well adsorbed from both aqueous acetic acid solutions and from nonaqueous solvents on a variety of pigment substrates. The deflocculating action of these and less soluble basic derivatives on finely divided pigments is due to hydrogen bonding between the basic groups and acidic hydrogen atoms in the film forming binders in the paint. This conclusion is supported by adsorption isotherms, cryoscopic, infra-red and deflocculation tests.

Studies on the haemoglobin of Arenicola marina

1. 1. Comparative studies on the purification of the oxyhaemoglobin from the blood of Arenicola marina by several methods show that ammonium sulphate precipitation causes marked denaturation of the chromoprotein but that effective purification is possible by absorption on calcium phosphate or chromatography on diethylaminoethyl cellulose. 2. 2. The absorption spectrum of the purified oxyhaemoglobin shows absorption bands at 574, 538 and 412–415 mμ. 3. 3. Reduction of the oxyhaemoglobin produces a mixture of products (including some unreduced oxyhaemoglobin) in which the true absorption maximum due to the haemoglobin cannot be determined. 4. 4. Arenicola oxyhaemoglobin forms a carboxyhaemoglobin and a nitric oxide haemoglobin which are very similar to the analogous derivatives of the mammalian respiratory pigment. 5. 5. Attempts to oxidize the iron to the ferric state causes denaturation of the invertebrate protein and the production of a product which is an atypical hemiglobin and which cannot be reduced to haemoglobin. 6. 6. The invertebrate pigment does not form a choleglobin or sulphaemoglobin. 7. 7. The absorption spectra, derivatives and behaviour during paper chromatography suggest that the same porphyrin is present in both Arenicola haemoglobin and mammalian haemoglobin. 8. 8. The prosthetic group-protein linkage in native Arenicola haemoglobin is broken under the identical conditions which cause the bond to split in the mammalian pigment. Breakage or modification of this linkage is one of the first results of mild oxidation of the invertebrate protein. 9. 9. The haemoglobin of Arenicola seems to contain one atom of iron and therefore one prosthetic group per Hufner unit of molecular weight 17,250.

The photoreduction of chlorophyll in the presence of diphosphopyridine nucleotide in pyridine solutions

1. 1. Both in the presence and absence of DPN, illumination with red light of oxygen-free solutions of chlorophyll a and ascorbic acid in aqueous pyridine led to the conversion: chlorophyll a (438 m μ) → photredued chlorophyll (420 m μ) → photoreduced pheophytin (390 m μ) → unknown derivative (370 m μ). The wavelengths in the parentheses are the positions of the absorption maxima of the substances. 2. 2. The presence of DPN (0.001 M) greatly reduced the rate of photoreduction of chlorophyll and altered the relative amounts of the pigment derivatives present after short periods of illumination, but did not change the nature of the reactions. 3. 3. The accumulation of DPNH in illuminated solutions of chlorophyll, ascorbic acid, and DPN was not confirmed. 4. 4. Previously reported data, which were attributed to DPNH accumulation, were probably due to different extents of conversion of chlorophyll in the presence and absence of DPN.

A survey of the antibiotic powers of yeasts.

SUMMARY: Examination of the antibiotic powers towards bacteria and moulds of 150 strains of yeasts from the British National Collection of Yeast Cultures has revealed that whereas none exhibited marked antibacterial properties certain of them inhibited the growth of moulds. Strains of Candida pulcherrima, when grown on a malt + yeast + peptone + glucose agar medium inhibited the growth of strains of certain Mucor, Fusarium and Penicillium spp. The inhibition was due to pulcherriminic acid, a derivative of the red pigment, pulcherrimin, formed by Candida pulcherrima. It inhibited growth of the moulds completely at 5000 p.p.m. and partially at 500 p.p.m.

血合筋肉の化学的研究-IX

Heat coagulability of myoglobin (Mb) and hemoglobin (Hb) of some fishes was compared under the conditions described below. Mb or Hb aq. solution was mixed with phosphate buffer to obtain a final concentration of pigment of 0.075%, and a final concentration and a final pH value of buffer, 0.2M and 6.7, respectively. These conditions were based on the results of preliminary experiments (cf. Figs. 1 and 2). The solution of various derivatives of pigment was prepared by ordinary methods, using 1 drop of 1% ferricyanide solution as oxidizing agent, 3mg. of solid KCN for cyanmet formation, and 3mg. of solid Na2S2O4 as reducer, per 5ml. of the solution. The subsequent procedure is essentially the same as that of BETKE7). Each 5ml. of the solution was pipetted into test tubes (16×ll0mm.), the tubes being stoppered with rubber stoppers and kept immersed in an ice-water bath. After heating at various temperatures in a water bath for 5min., each tube was immersed immediately in the ice-water bath again, and the concentration of pigment in the supernatant determined spectrophotometrically. Then a curve (termed heat coagulation curve) is obtained by plotting the remaining amounts of pig-ment against the temperatures. The heat coagulation curves of both pigments are shown in Fig. 3. The noticeable points of these results are as follows: (1) The positions of heat coagulation curves were different among species, Mb and Hb, and derivatives, and the slope of curve of Mb was generally steeper than that of Hb. (2) On both Mb and Hb, met form was the most heat sensitive, followed by cyanmet form, while carbonyl and reduced forms were comparatively stable. (3) Each derivative of Hb in a species coagulated at far lower temperatures than the cor-responding one of Mb, and this tendency was marked in met and cyanmet forms. (4) Species specificity of Mb in heat coagulability was fairly evident, which makes it possi-ble to divide the fishes tested into two groups; a group having heat resistant Mb such as tuna, bonito, and swordfish, and another group having heat irresistible Mb such as yellowtail, saury-pike, and mackerel. Both Mb and Hb of horse, studied for reference, were found to be very heat resistant as compared with the pigments of fish. (5) Under the conditions employed, however, there was a limit of temperature beyond which no further coagulation of pigment was measured, in the case of Mb of bonito, swordfish, and horse.

Cytochrome system in Bacterium coli commune

THE aerobic respiration of Bact. coli, like that of Bacillus subtilis, baker's yeast and other aerobic cells, is strongly and reversibly inhibited by KCN, H2S, NaN3 and CO, but, unlike that of B. subtilis or cells of baker's yeast, the inhibition with CO is not light-sensitive. The absorption spectrum of cytochrome in B. coli differs markedly from that of B. subtilis or yeast cells. Thus,,the band a is absent from B. coli, and is replaced by a hardly perceptible shading a, at 590 m,u and a feeble band a2 at 628 mp, which is similar to the band a2 of Azotobacter. This band in organisms exposed to CO moves to 634 m,u, on oxidation it is shifted to 645 mp, and in presence of KCN and 02 it disappears completelyl [Keilin, 1934; Fugita & Kodama, 1934]. The band a2 belongs therefore to an autoxidizable haemochromogen compound which reacts with KCN and with CO. According to Lemberg & Wyndham [1937] this compound can be considered as a bile pigment haemochromogen with -an open tetrapyrrolic chain kept together in a ring form by the central iron atom. Lemberg ascribes to a2 the structure of a biliviolin-haemochromogen which is obtained by dehydrogenation of verdohaemochromogen. The component a2 appears therefore to belong to a bile pigment derivative of haematin a, and, as in all the derivatives with an open tetrapyrrolic chain [Holden & Lemberg, 1939], its absorption spectrum is devoid of the Soret or y-band. The properties of a2., namely its reactions with 02, C0 and KCN, suggest that in B. coli, as in Azotobacter [Negelein & Garischer, 1934], this component may act as cytochrome oxidase. On the other hand, the failure to find any parallelism between the concentration of a2 and the respiratory activity of cells containing this component leaves open the possibility of the existence in B. coli of yet another autoxidizable haematin compound acting as cytochrome oxidase and capable of oxidizing the component b,. The bands b and ; of a typical cytochrome are replaced in B. coli by one band which lies at about 560 nip and which represents the oc-band of a non-autoxidizable haemochromogen compound bl. This band is asymmetric and shows on its short-wave side a reinforcement which, according to Yamagutchi [1937], may represent the band c masked by an extension of the band bl. On freezing and cooling the suspension of B. coli to the temperature of liquid air, the short-wave side of the band b, becomes greatly intensified and appears at 551 m, united by a shading to a much fainter band at 559 m,. The oc-band of cytochrome c which, under similar conditions, is also markedly intensified is, however, shifted from ita usual position at 649-5 to 547 m,. This clearly shows, even if we assume that the asymmetric band b, of B. coli is composed of two

Further Observations on the Red Pigments of Pellagra Urines

Conclusions1. The color noted in positive Beckh-Ellinger-Spies tests is not due to porphyrin. While a marked increase in urinary porphyrin, such as occurs in porphyria, would be productive of color, the color reaction as observed in pellagra and other diseases, is due to urorosein, first described by Nencki and Sieber. There is no evidence that the color reaction is due to any bile pigment derivative. 2. The urines of pellagra patients may contain either the chromogen of urorosein, or a red pigment extracted by the toluene preservative. It appears highly probable that this pigment is indirubin, although exact identification has not yet been made. 3. Both of the red substances may be noted in the urines of patients not having clinical pellagra. Further investigation is necessary to decide whether their appearance is related to deficiency of nicotinic acid or other essential substances.

The chemical nature of hæmochromogen and its carbon monoxide compound

In a previous communication (Hill, R., 1925) I summarised the properties of a number of derivatives of the blood pigment in which the iron was replaced by different metals. When iron combines with hæmatoporphyrin to give a hæmatin, the spectrum resembles that of a typical metallo-porphyrin only when the pigment is in the form of hæmochromogen. The nature of hæmochromogen has until quite recently not been understood. The work of Anson and Mirsky (1925) shows that hæmochromogen, prepared directly from oxyhæmoglobin by reduction in alkaline solution, is a compound between the pigment and the protein. Again, these workers state clearly that when other simpler substances are used instead of the protein, each substance gives its characteristic hæmochromogen by actual combination. They emphasised the fact that all substances capable of yielding typical hæmochromogens contained nitrogen, and showed that hæmochromogen is dissociated in solution into pigment and free nitrogen compound, to a greater or less extent depending on the particular hæmochromogen in question. Dilling (1910) had made a detailed qualitative study of hæmochromogens prepared by means of different reagents, but he does not seem clear as to the part played by the reduced hæmatin in the formation of hæmochromogen. The nearest approach to a solution of the problem previous to Anson and Mirsky’s work was that of Bertin Sans and de Moitessier (1893). These authors had shown that pure hæmatin when reduced with sodium hydrosulphite will not give hæmochromogen, unless albumen or certain amines were added. They failed, however, to exclude the possibility of the relation between pigment and nitrogenous substance being other than chemical combination. Von Zeynek (1910) analysed pyridine hæmochromogen and found that it contained 2·2 molecules of pyridine. He regarded it as a compound of hæmochromogen and pyridine. This work, therefore, gave no indication of the minimum amount of pyridine necessary to convert reduced hæmatin into hæmochromogen.

THE UROBILIN PROBLEM

It is nearly half a century since urobilin was described by Jaffe. Little attention was paid to this substance from a clinical standpoint until its relation to the decomposition of bile pigment by bacterial action in the alimentary canal was made prominent by Friedrich Müller. The terms "urobilin" and "urobilinogen" are applied to a group of derivatives of bile or blood pigment for which methods of estimation, crude though they may appear to careful analysts, have been devised and widely applied to the examination of urine and feces by a considerable number of clinicians and in a great diversity of cases. Like many novelties in clinical chemistry—we may cite the indican test and Ehrlich's diazoreaction—the test for urobilin has attained a considerable popularity and has been the subject of generalizations that might appear highly significant and diagnostic to the uncritical reviewer. It seems worth while, therefore, to point out at