Quantitative determination of monosubstituted guanidines: A comparative study of different procedures

Analysis of N²-acyl arginine derivatives as well as of arginine analogs lacking in a α-amino function by Weber's modification of the Sakaguchi procedure yielded colored complexes with absorbance values approximately twice that obtained with an equivalent concentration of unmodified arginine. The limitations concerning the applicability of the various modification of the Sakaguchi procedure as well as of fluorimetric assay to the quantitative estimation of a variety of monosubstituted guanidines and proteins are discussed.     

Properties of the olfactorymucosa's water-soluble proteins

A quantitative and qualitative biochemical investigation was carried out for the composition of water-soluble proteins in preparation of the bull's olfactory mucosa scrape. Comparing the results with those obtained for a similar preparation of a nonsensory nature (a preparation of a respiratory epithelium scrape) the authors found that the most electrically-mobile protein fractions differ in their physical properties and number. A reaction for glycoproteins showed that with an alcian blue an appropriate colour for acidic glycoproteins is given only by two protein fractions. The most electrically-mobile fraction with its visual colour of an olfactory pigment is probably identical to its water-soluble component absorbed on phosphatides. The second, weakly mobile protein is the main acidic glycoprotein of the olfactory mucus and besides it has a reaction for on alkaline phosphatase.     

Comparative microphotometric studies of DNA and arginine in plant nuclei

Summary Photometric measurements have been made of the amounts of stain formed in the Feulgen (DNA) and Sakaguchi (arginine) reactions in plant nuclei of differing ploidy.In nuclei of diploid and tetraploid plants of Tradescantia ohioensis and of diploid, triploid and tetraploid plants of Ranunculus ficaria, both Feulgen and Sakaguchi values gave ratios which agreed closely with the ratios of the number of chromatids known to be present. The Feulgen/ Sakaguchi ratio for each of the different types of nuclei measured was very similar both within and between these two species.In the interphase nuclei of five different species, both Feulgen and Sakaguchi values gave bimodal distributions. In the nuclei of differentiating cells, the proportions of values falling into each of the 2C, 4C or 8C classes were the same for both stains.Measurements of the amounts of both stains were made in sequence on the same individual nuclei and a positive correlation found between the two sets of values.In nuclei from differentiating cells of Vicia faba primary roots, the Feulgen/Sakaguchi ratio decreased with increasing distance from the apex.The following suggestions were made from the results: (a) that there is some degree of quantitative constancy of nuclear arginine which parallels that of DNA; (b) that the amount of nuclear arginine, like that of DNA, is doubled during synthesis in interphase; (c) that the syntheses of DNA and arginine in interphase, if not simultaneous, at least occur within the same relatively short period; (d) that there may be a difference in the DNA/arginine ratio between the nuclei of meristematic and differentiating cells.     

Deamination by the diazotization-deazotization method

Synopsis The diazotization-deazotization method for deaminating proteins in fixed tissue section, as originally described by Stoward (1963), has been investigated further with the following results. Blocking of arginine was confirmed by the Sakaguchi method and with anionic dyes. What appeared to be staining of lysine by anionic dyes was blocked but the dinitrofluorobenzene reaction was not. Blocking of histidine could not be demonstrated. Tyrosine was blocked to Millon's reagent but not to the coupled tetrazonium reaction. Staining of sulphydryl groups by Barrnett & Seligman's dihydroxydinaphthyldisulphide method was reduced but not obliterated. The colour of the reaction product between tryptophan residues and dimethylaminobenzaldehydenitrite was altered. Explanations for these findings are suggested.     

Protein-polymer grafts. III

Arginines found on protein backbone can be modified easily, using 10 molar excess of 4-hydroxybenzil in 70% ethanolic alkali solution (0.25M), at room temperature. 96% of the arginines are modified this way, as measured by Sakaguchi reaction. The phenolic hydroxide was available for an enzyme, tyrosinase, to be oxidized.Second paper of this series, this journal pp. 63–68.     

A colorimetric micro method for the determination of formyl groups

The characteristic purple colour formed by N-formyl-N'-2,4-dinitrophenyl-hydrazine in the presence of piperidine and acetone was made the basis of a new quantitative method for the determination of formyl groups. Samples containing N-formyl groups (up to 0.4mumole) are hydrazinolysed at 97-98 degrees for 1hr. and are dinitrophenylated after the removal of excess of hydrazine. Interference from 2,4-dinitrophenylhydrazine is eliminated by subjecting the dinitrophenylated samples to chromatography on an alumina column. Interference arising from the formation of N-acetyl-N'-2,4-dinitrophenylhydrazine, when determining formyl groups in samples containing acetyl, can be avoided by a paper-chromatographic separation before analysis. A standard procedure is described. The method gives satisfactory results when applied to N-formyl-amino acids. Gramicidin, when analysed by this method, was found to contain 0.89 mole of formyl group/mole for a molecular weight of 1880. The method indicated the absence of formyl groups from lysozyme, a protein known not to contain such groups. Generally, the analytical values obtained by the method are within 100+/-4% of theory.     

Colour change: A novel biomarker indicating sublethal stress in the millipede Julus scandinavius (Diplopoda)

Abstract

During investigations on the induction of the 70 kD stress protein family (hsp70, stress-70) in Julus scandinavius following exposure to different biohazards, the colour of the supernatant of the homogenate was closely correlated to the hsp70 level. Hsp70 has recently been shown to be a suitable biomarker for sublethal toxicity in soil animals. Control millipedes typically exhibited red or red-orange supernatants whilst the supernatant of starved or toxin-exposed diplopods was orange, orange-yellow, or even bright yellow. Based on these observations, a quantitative colour test was established which was found to be able to indicate the degree of stress situations caused by exposure to heavy metals (cadmium, zinc), organic pollutants (lindane, PCB 52), or by food deprivation in laboratory tests. It is suggested that this is caused by a breakdown of the red-orange bilirubins into orange-yellow urobilins.     

Side effects of reaction media in histochemical blocking procedures

Synopsis 0.2 N NaOH, the reaction medium for 1,2-cyclohexanedione, a specific reagent for arginyl residues in proteins, was found to intensify, at some sites in rat abdominal skin and human gingiva, the Sakaguchi reaction, staining with Pauly's reagent, and anionic dye binding at pH 6.4; at other sites these reactions were reduced, presumably due to extraction of material from sections. 0.2 N NaOH slightly reduced staining after the ninhydrin-Schiff procedure at all sites in rat skin. The interpretation of this finding is obscure, because some sites giving a positive Sakaguchi reaction and staining with anionic dyes failed to stain after the ninhydrin-Schiff procedure. There were also alterations in staining, with the cationic dyes Alcian Blue and Alcian Yellow. It is suggested that 0.2 N NaOH ruptures linkages between polycationic residues of proteins and polanions, demonstrable by Alican Blue. The blockade produced by acetic anhydride-pyridine (4060 v/v) mixtures was stable, in the alkaline conditions required for staining with Pauly's reagent. Pretreatment with pyridine alone reduced tissue binding of anionic dyes.     

The nature of the seasonal colour dimorphism in the scorpion fly,Panorpa japonica thunberg

The scorpion fly, Panorpa japonica, displays a seasonal colour dimorphism by changing from black to yellow in the adult state. The yellow pigment in the integument and haemolymph of the adult fly was identified as sepiapterin, while the black integument pigment was found to be melanin. After analysis of sepiapterin content by high performance liquid chromatography and determination of total haemolymph volume by [carboxyl-¹⁴C]inulin, it was shown that sepiapterin levels in the haemolymph and integument varied greatly both in the two colour types of insects and in the two sexes. Photometric analysis of melanin content showed that melanin levels correlated negatively with sepiapterin levels. These quantitative differences in sepiapterin and melanin fully explain the colour dimorphism in the insect.     

The additional Sakaguchi positive component in chymotrypsin A alpha.

Quantitative Sakaguchi analysis of the B- and C- chains of chymotrypsin revealed the presence, in the latter chain, of an additional Sakaguchi positive component which was not detectable by amino acid analysis using ion-exchange chromatography. Analysis of the cyanogen bromide cleavage products of the C-chain suggests that the peptide sequence 149–192 of chymotrypsin is responsible for the additional Sakaguchi positive component.     

Improved borate method for the rapid distinction of glucosamine and galactosamine in an exopolysaccharide produced by Citrobacter sp

An improved borate method for the quantitative distinction of glucosamine (GlcN) and galactosamine (GalN) in a mixture is presented which is based on the Elson-Morgan method with addition of sodium borate to differentiate colour formation by the two hexosamines. The r2 value and maximum deviation of the method based on calculations derived in this study were 0.9979 and 5.1 %, respectively. Using this method, the GlcN/GalN ratio in an exopolysaccharide (EPS) produced by Citrobacter sp. was found to change with time during the production process, with a maximum value at 9.8:1.     

Effects of anhydrous benzoylation on staining with a variety of protein end-group methods

Summary The effects of anhydrous benzoylation were examined in connection with the following protein end-group methods: diazosulfanilic acid — Azure A, the Sakaguchi reaction and a fluorescent variant, the phenanthrequinone fluorescent method for arginine, the Millon reaction, the Morel-Sisley reaction, the mercury orange method, the alloxan and ninhydrinShiff methods, the Dansyl-chloride fluorescent method for lysine, the dimethylaminobenz-aldehydenitrite (DMAB) method for tryptophan, and the acid fluorochrome brilliant sulfoflavin used at pH 2.8 as a stain for basic groups. With all of these methods except the DMAB method for tryptophan, selective blockage of cytoplasmic proteins and staining of nuclei was obtained. With the DMAB reaction, the results were reversed: cytoplasmic staining exceeded nuclear staining after benzoylation.     

Environmentally induced changes in carotenoid-based coloration of female lizards: a comment on Vercken et al

Colouration may either reflect a discrete polymorphism potentially related to life-history strategies, a continuous signal related to individual quality or a combination of both. Recently, Vercken et al. [J. Evol. Biol. (2007) 221] proposed three discrete ventral colour morphs in female common lizards, Lacerta vivipara, and suggested that they reflect alternative reproductive strategies. Here, we provide a quantitative assessment of the phenotypic distribution and determinants of the proposed colour polymorphism. Based on reflectance spectra, we found no evidence for three distinct visual colour classes, but observed continuous variation in colour from pale yellow to orange. Based on a 2-year experiment, we also provide evidence for reversible colour plasticity in response to a manipulation of the adult population sex ratio; yet, a significant portion of the colour variation was invariant throughout an adult female's life. Our results are thus in agreement with continuous colour variation in adults determined by environmental factors and potentially also by genetic factors.     

Studies on middle silkgland proteins of cocoon colour sex-limited silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis> L.) using two-dimensional polyacrylamide gel electrophoresis

Qualitative and quantitative differences in proteins expressed in the middle silkglands of male and female silkworm larvae that differ in silk colour were investigated by high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer assisted image analysis. About 1000 protein spots were resolved in both the sexes and most proteins were shown to be distributed in the area from 15 kDa to 70 kDa and pH 4–8. It was found that some proteins displayed higher expression in yellow cocoon, while two proteins were only expressed in female silkworm silkgland tissue through the comparison and analysis by two-D software. These proteins especially existed in female silkworm middle silkgland tissue of yellow cocoon. Furthermore, these proteins might be involved in the expression of cocoon colour phenotype     

Reactivity of consecutive basic amino acid residues in peptides

Different tetrapeptides of general formula L-Ala-X-X-Gly, possessing a basic doublet in the second and third position (X = Arg or Lys), have been synthesized as free or N-acetylated molecules. The chemical reactivity of the arginine guanidino group and of the lysine epsilon-amino group were studied using respectively the Sakaguchi and the ortho-diacetylbenzene reactions, in the tetrapeptides as well as in related molecules. In both cases, the colour yield is markedly influenced by the length of the polypeptide chain and by the relative positions of the arginine and lysine residues, suggesting the occurrence of intramolecular bonds within the tetrapeptide molecule. Tryptic hydrolysis of the tetrapeptides was followed by evaluating the amino acids or peptides which appear to be specific for the different possible cleavages at the arginyl or at the lysyl bonds. The susceptibility to trypsin of the carboxylic group of the second basic amino acid decreases progressively in the order Lys-Arg greater than Arg-Arg much greater than Lys-Lys greater than Arg-Lys, which shows a fair correlation with the intra-cellular cleavage of the bonds observed during the processing of preproteins of of the precursors of several physiologically active peptides.     

Single nucleotide polymorphisms in the Melanocortin 1 Receptor gene are linked with lightness of fibre colour in Peruvian Alpaca (Vicugna pacos)

Melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) (MC1R) is a gene‐controlling melanogenesis in mammals. However, it is not well characterized in alpacas and its association with colour is not known. The aim of this study was to look for polymorphisms in the MC1R gene in Peruvian Huacaya alpacas and to analyse the relationship between MC1R single nucleotide polymorphisms (SNPs) and the variations in the instrumental measurement of colour of alpaca fibre. Sixty alpaca fibre samples from black, brown, cream and white animals (15 for each colour) were used to extract DNA from hair bulbs. Colour was measured with a spectrophotometer to obtain quantitative values (CieL*a*b*). Sixteen samples, four of each colour group, were sequenced. Eighteen SNP mutations, 10 not previously described, were found in these 16 sequences. Three of them were chosen (c.82A>G, c.865C>T, c.901C>T) to analyse genotypes by PCR‐RFLP in the other 44 fibre samples and to determine the association of mutations with instrumental colour. These three polymorphisms showed association with fibre lightness (P < 0.05), although there was no correlation with colour groups.     

Contemporary divergence of island bird plumage

Although the diversity in avian plumage coloration is striking, there is little known about the rate with which colour diverges. Eastern bluebirds Sialia sialis bermudensis on the island of Bermuda are considered endemic based upon differences in coloration from the mainland, but recent molecular evidence suggests they established on the island only 400 yr ago. We explored sexual dichromatism and colour divergence in this isolated population, thus providing one of the few quantitative accounts of contemporary plumage change. Contrary to expectations that sexual dichromatism would decrease in this sedentary island population, we found that males and females have increased plumage ornamentation in a coordinated fashion that acts to preserve sexual dichromatism, while plumage colour is also altered to become brighter and bluer. These differences were in place at least 100 yr ago based upon a separate analysis of museum specimens. Our results provide insight into the divergence of plumage colour in an incipient species, and we show the remarkable extent to which plumage colour can change over contemporary time frames.     

Stimuli eliciting courtship from domesticated zebra finches

Previously it has been suggested that both the beak colour and the behaviour of the female zebra finch affect the expression of male courtship. Experiments were carried out to allow identification of the major stimuli eliciting singing, pivoting and beak wiping from males. They were found to be behaviour, beak colour, and behaviour respectively. We also present quantitative evidence indicating a positive feedback between male singing and female bowing; this is consistent with a previous speculation that bowing is a solicitation signal to males.