[38] The rapid determination of amino groups with TNBS

Fields, Robert

doi:10.1016/s0076-6879(72)25042-x

Cited by 461 publications

(225 citation statements)

References 15 publications

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“…The free amino groups of ethylenediamine spacers introduced into the PVA molecule were measured by the 2,4,6-trinitrobenzenesulfonic acid (TNBS) method. [21][22][23] One hundred microliters of DMSO containing 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (0.27 mg) and N-hydroxysuccinimide (NHS) (0.16 mg) were added to 1 mg of the ADOX-isomers dissolved in 0.1 ml of DMSO, respectively, followed by stirring for 2 h at room temperature. Then, PVA-ethylenediamine (10 mg) dissolved in 0.2 ml DMSO was added to the activated-ADOX-isomers and stirred for 4 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

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Synthesis of Poly(vinyl alcohol)-Doxorubicin Conjugates Containing cis-Aconityl Acid-Cleavable Bond and Its Isomer Dependent Doxorubicin Release

Kakinoki

Kaneo

Ikeda

et al. 2008

Biological & Pharmaceutical Bulletin

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Aconityl-doxorubicin (ADOX) was synthesized by the modified method of Shen and Ryser. Two isomers of cis-ADOX (cis-configuration) and trans-ADOX (trans-configuration) were generated in the reaction of DOX and cis-aconitic anhydride. These products were separated completely by using HPLC and analyzed by TOF-MS spectroscopy and 1 H-and 13 C-NMR experiments. The yields of cis-ADOX and trans-ADOX were 36.3 and 44.8%, respectively. The free g g-carboxylic group of ADOX molecule was coupled to poly(vinyl alcohol) (PVA) via ethylenediamine spacer, resulting the macromolecular conjugates of PVA-cis-ADOX and PVA-trans-ADOX, respectively. The DOX content of the conjugates estimated by the hydrolysis method detected the aglycone of DOX which can be estimated as the PVA-bound DOX selectively was 4.4 w/w% which was similar to 4.6 w/w% by the ordinary UV method. Both PVA-cis-ADOX and PVA-trans-ADOX were very stable at neutral pH, but the release of DOX was increased markedly under acidic conditions. Half-life of the release of DOX from PVA-cis-ADOX at pH 5.0 was 3 h which was 4.7-fold shorter than that from PVA-trans-ADOX (14 h). The cytotoxicities of PVA-cis-ADOX and PVA-trans-ADOX were evaluated by using J774.1 cells employing a [ 3 H]uridine incorporation assay as a measure of RNA synthesis. A significant difference in antitumor activity between PVA-cis-ADOX and PVA-trans-ADOX was observed where the former was much active than the later. It was suggested that the conjugate enters the cells and reaches the lysosomal/endosomal compartment, and that the aconityl spacer releases DOX from the conjugate in the acidic compartment of lysosomes/endosomes due to the participation of a free carboxylic group.

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Section: Methodsmentioning

confidence: 99%

“…The number of free amino groups of the PVA-ethylenediamine was estimated by the TNBS method. [21][22][23] One mole of PVA showed the color intensity of 25 mol of free amino group. Free g-carboxylic group of ADOX molecule was activated with an equimolar amount of EDC and NHS, and then reacted with PVA-ethylenediamine.…”

Section: Synthesis Of Aconityl-doxorubicinmentioning

confidence: 99%

Synthesis of Poly(vinyl alcohol)-Doxorubicin Conjugates Containing cis-Aconityl Acid-Cleavable Bond and Its Isomer Dependent Doxorubicin Release

Kakinoki

Kaneo

Ikeda

et al. 2008

Biological & Pharmaceutical Bulletin

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“…The free amino groups were determined using 2,4,6-trinitrobenzenesulfonic acid(TNBS), following the method of Fields (1972). Briefly, fish gelatin (0.2 g) was dissolved in 10 mL of phosphate buffer (pH 8.2).…”

Section: Free Amino Group Determinationmentioning

confidence: 99%

Physico-chemical properties of gelatin from bighead carp (Hypophthalmichthys nobilis) scales by ultrasound-assisted extraction

et al. 2013

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In this study, gelatin was extracted from bighead carp (Hypophthalmichthys nobilis) scales by water bath (WB) and ultrasound bath (UB) at 60°C for 1 h, 3 h and 5 h, named WB1, WB3, WB5, UB1, UB3 and UB5, respectively. The physicochemical properties of gelatin were investigated. The result indicated that gelatin extracted from bighead carp scales had a high protein content (84.15~91.85 %) with moisture (7.111 3.65 %), low ash content (0.31~0.97 %). All extracted gelatin contained α-and β-chains as the predominant components. Gelatin extracted by UB obtained much higher yield (30.94-46.67 %) than that of WB (19.15-36.39 %). More voids and less sheets of gelatin structure were observed, when the gelatin was extracted by UB for longer time. Gelatin of UB-assisted extraction normally exhibited lower gel strength and melting points than that of WB, which may be resulted from the protein degradation reflected by the FTIR spectra and higher free amino group content. However, there was no significant difference between WB1 and UB1. Therefore, the ultrasound assisted extraction in a short time was a promising method to enhance the yield and obtain gelatin with high quality.

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“…A farinha e frações protéicas isoladas foram hidrolisadas pelo uso de solução de pepsina (SIGMA P-7012), por três horas, procedimento seguido de ajuste de pH, adição de solução de pancreatina (SIGMA P-1750) e banho a 37 °C por 24 h. A reação foi interrompida pela adição de ácido tricloroacético (TCA) 10%. A percentagem de hidrólise foi determinada pela medida dos novos aminoácidos livres gerados, quantificando-se o número de grupamentos amino-livres no sobrenadante, pela reação com reagente TNBS, conforme descrito por FIELDS [21] e modificado por SPADARO et al [49]. A digestibilidade foi avaliada após cálculo do grau de hidrólise, tomando-se a caseína (SIGMA C-8654) como padrão (100%).…”

Section: -Digestibilidade In Vitrounclassified

Isolamento da globulina majoritária, digestibilidade in vivo e in vitro das proteínas do tremoço-doce (Lupinus albus L.), var. Multolupa

Neves

Silva

2006

Ciênc. Tecnol. Aliment.

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RESUMOOs objetivos do trabalho foram isolar, purificar e estudar algumas propriedades da fração globulina majoritária de tremoço-doce, var. Multolupa; assim como avaliar as características de digestibilidade da farinha e frações isoladas. As frações protéicas foram separadas por fracionamento diferencial com uso de diferentes solventes. A globulina majoritária de tremoço-doce foi isolada, purificada por cromatografia em Q-Sepharose, revelando um único pico de proteína. Apresentou um peso molecular de 162,5 ± 10,0 kDa, determinado por cromatografia em Sephacryl S-300, e subunidades entre 20-70 kDa em PAGE-SDS. A solubilidade em função do pH e concentrações de NaCl revelaram curva típica dessa fração. A digestibilidade da proteína da farinha e das frações albumina, globulina e glutelina foi avaliada por experimentos in vitro e in vivo e se revelou alta para a fração globulina, seguida pela glutelina, albumina e farinha. A digestibilidade in vivo da fração globulina, tanto aparente quanto verdadeira, não diferiu significativamente daquela determinada para a caseína. Apesar da alta digestibilidade da fração majoritária e frações protéicas, a utilização como única fonte de proteína nas dietas revelou valores baixos de RPLR (razão protéica líquida relativa), indicando ser insuficiente para sustentar o crescimento dos animais, comparativamente à caseína. Palavras-chave: Lupinus albus, frações protéicas, globulina principal, digestibilidade in vitro e in vivo, ensaio biológico. SUMMARY ISOLATION OF MAJOR GLOBULIN, IN VIVO AND IN VITRO DIGESTIBILITY OF PROTEINS FROM SWEET LUPIN (Lupinus albus L.)VAR. MULTOLUPA. The aim of the present work was to isolate, purify and study some properties of the major globulin fraction from sweet lupin, var. Multolupa; as well as to evaluate the digestibility features of flour and isolated fractions. The protein fractions were separated by differential fractionation with different solvents. The lupin major globulin was isolated, purified by Q-Sepharose chromatography when it showed only one protein peak. The major protein presented a molecular weight of 162.5 ± 10.0 kDa, determined on Sephacryl S-300 chromatography; and subunits between 20 to 70 kDa on SDS-PAGE. The protein solubility in relation to pH and NaCl concentrations showed a typical S-like curve for globulins. The flour and the isolated protein fractions, albumins, globulins and glutelins were evaluated by in vitro and in vivo experiments. The in vitro and in vivo digestibility assays revealed a high digestibility for the globulin, followed by glutelin, albumin and the flour. The globulin in vivo digestibility did not differ significantly from that obtained to casein. Despite the high digestibility encountered for the major protein fraction and protein fractions, the use as the only protein source in the diets revealed low values for RNPR (relative net protein ratio), showing it to be insufficient in terms of sustaining the growth of animals, comparatively to casein. Keywords: Lupinus albus, protein fractions, major globulin, in ...

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[38] The rapid determination of amino groups with TNBS

Cited by 461 publications

References 15 publications

Synthesis of Poly(vinyl alcohol)-Doxorubicin Conjugates Containing cis-Aconityl Acid-Cleavable Bond and Its Isomer Dependent Doxorubicin Release

Synthesis of Poly(vinyl alcohol)-Doxorubicin Conjugates Containing cis-Aconityl Acid-Cleavable Bond and Its Isomer Dependent Doxorubicin Release

Physico-chemical properties of gelatin from bighead carp (Hypophthalmichthys nobilis) scales by ultrasound-assisted extraction

Isolamento da globulina majoritária, digestibilidade in vivo e in vitro das proteínas do tremoço-doce (Lupinus albus L.), var. Multolupa

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