The measurement of amino groups in proteins and peptides

Fields, Robert

doi:10.1042/bj1240581

Cited by 410 publications

(149 citation statements)

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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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“…Therefore, only the fraction of free lysine amino-groups in proteins that were modified with charge-bearing groups was quantified, using trinitrobenzenesulphonic acid titration method (Fields, 1971). We found that approximately 25-35% of all aminogroups accessible for modification was modified with chargebearing groups.…”

Section: Measurement Of Molecular Charge and Weightmentioning

confidence: 97%

True

et al. 2000

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Summary Molecular charge is one of the main determinants of transvascular transport. There are, however, no data available on the effect of molecular charge on microvascular permeability of macromolecules in solid tumours. To this end, we measured tumour microvascular permeability to different proteins having similar size but different charge. Measurements were performed in the human colon adenocarcinoma LS174T transplanted in transparent dorsal skinfold chambers in severe combined immunodeficient (SCID) mice. Bovine serum albumin (BSA) and IgG were fluorescently labelled and were either cationized by conjugation with hexamethylenediamine or anionized by succinylation. The molecules were injected i.v. and the fluorescence in tumour tissue was quantified by intravital fluorescence microscopy. The fluorescence intensity and pharmacokinetic data were used to calculate the microvascular permeability. We found that tumour vascular permeability of cationized BSA (pI-range: 8.6-9.1) and IgG (pI: 8.6-9.3) was more than two-fold higher (4.25 and 4.65 × 10 -7 cm s -1 ) than that of the anionized BSA (pI ≈ 2.0) and IgG (pI: 3.0-3.9; 1.11 and 1.93 × 10 -7 cm s -1 , respectively). Our results indicate that positively charged molecules extravasate faster in solid tumours compared to the similar-sized compounds with neutral or negative charges. However, the plasma clearance of cationic molecules was ~2 × faster than that of anionic ones, indicating that the modification of proteins enhances drug delivery to normal organs as well. Therefore, caution should be exercised when such a strategy is used to improve drug and gene delivery to solid tumours.

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“…For example, conventional colorimetric (TNBS, fluorometric) 21,22 and chromatographic characterization (HPLC-SEC, RP-HPLC, gel permeation chromatography) [23][24][25] in combination with light scattering, MALDI-MS 24,[26][27][28] , and capillary electrophoresis 29,30 have been applied for analysis of PEGylated proteins. Specific colorimetric detection of PEG with iodine 31, 32 using Fourier transform infrared (FTIR) or Raman spectroscopy 24 and nuclear magnetic resonance 33, 34 have also been described.…”

Section: Introductionmentioning

confidence: 99%