Carrying Amino Groups Research Articles

Topological analysis of components of the cytochrome b6f complex by chemical crosslinking

The cytochrome b 6 f -complex from spinach thylakoids has been chemically crosslinked with DSP (3,3′-dithiobis(succinimidylpropionate)), DTBP (dimethyl-3,3′-dithiobis(propionimidate)) and SADP ( N -succinimidyl[(4-azidophenyl)dithio]propionate). By two-dimensional diagonal SDS-polyacrylamide gel-electrophoresis the following crosslinks were identified: cytochrome f -subunit IV, cytochrome b -subunit IV and cytochrome f -cytochrome b . Diepoxybutane did not yield crosslinks. These results suggest that protein domains carrying amino groups are spaced apart for these membrane proteins in a distance of 10–15 A.

Hinge-thiol coupling of monoclonal antibody to silanized iron oxide particles and evaluation of magnetic cell depletion

Iron oxide particles of average size 0.5-1.5 microns, covered by a silane coat carrying amino groups (Bio-Mag, Advanced Magnetics, Boston), were derivatized by reaction with N-[(gamma-maleimidobutyryl)oxy]-succinimide (GMBS), N-hydroxysuccinimidyl iodoacetate (NHIA), 2-iminothiolane (2-It), or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The derivatized particles were suitable for the reaction with sulfhydryl groups and subsequently coated with monoclonal antibodies (MoAbs) of different classes and isotypes (IgM, IgG1, IgG2a, IgG2b, IgG3) as well as polyclonal rabbit anti-mouse IgG (RAM). The antibodies were reduced by dithiothreitol (DTT) and covalently conjugated to the BioMag derivatives via liberated sulfhydryls of the hinge region. The observed conjugation ratios, expressed as protein/iron (micrograms/mg), could be reproducibly varied for optimization. These ratios were dependent on the type and amount of antibody offered for coupling to the derivatized particles, decreasing as follows: polyclonal = IgM greater than IgG2b greater than IgG2a = IgG3 greater IgG1. The conjugation ratios were also dependent on the type and amount of the spacer used to derivatize the BioMag particles, decreasing as follows: GMBS greater than NHIA greater than 2-It greater than SPDP. The magnetically responsive magnetite-antibody conjugates ("magneto-beads"), carrying MoAb BMA 081 (anti-CD8; IgG2a), MoAb BB10 (anti-CD10/CALLA; IgG2b), MoAb VIL-A1 (anti-CD10; IgM), and polyclonal RAM, coupled similarly via 3.6 mumol of GMBS spacer per mg of Fe, were further investigated with respect to a depletion effect on specific cell subsets. The rates of cell depletion were found to be strongly dependent on the individual characteristics of the antibody used.(ABSTRACT TRUNCATED AT 250 WORDS)

Funktionalisierung des perlcopolymerisationsprodukts aus methylacrylat und 1,4‐bis(vinyloxy)butan

AbstractThe ester groups of copolymer beads, prepared from methyl acrylate and 1,4‐bis(vinyloxy)‐butane, were substituted by aminolysis with hexamethylenediamine or aminoalcohols. The resulting gel carrying amino groups was transferred into an anion exchanger by alkylation with 2‐chloroethanol or into a cation exchanger by reaction with succinic anhydride. Hydrazinolysis of the starting copolymer beads resulted in gels with hydrazide groups which subsequently can be derivatized. Gels with hydroxyl groups linked to the matrix by means of spacers were obtained by reaction of the succinylated gels with 2‐aminoethanol. Phosphorylation of these hydroxyl groups afforded strongly acidic cation exchangers. The yields of the derivatized products are higher and the preparations are easier than those of the known methods. The 11 gel derivatives synthesized from the starting gel are mechanically stable, resistent to hydrolysis in the pH range between 1 and 14, chemically stable at temperatures up to 130°C and obtainable with a variety of pore sizes. The derivatized, cross‐linked polymer beads, applicable in aqueous systems as well as in organic solvents, should find application in chromatography, in solid‐phase synthesis and as basic gels in biotechnical processes.