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N-Hydroxysuccinimide Carbonates and Carbamates are Useful Reactive Reagents for Coupling Ligands to Lysines of Proteins
No full textLigands containing amino or hydroxyl groups were converted to their corresponding activated N-hydroxysuccinimidyl carbamate and carbonate by reaction with disuccinimidyl carbonate (DSC). The latter reagents can be used for the group-specific modification of primary amines as an alternative to the widespread usage of N-hydroxysuccinimide esters. Biotin and 2,4-dinitrophenyl (DNP) derivatives were used as examples to demonstrate the approach. Biotin and DNP were each extended by attaching two different spacer arms, carrying either a hydroxyl group or a primary amine as terminal functions. The latter were then activated via their conversion to N-hydroxysuccinimide carbonates and carbamates, respectively. The usefulness of these reagents for protein modification was investigated. The modified proteins obtained exhibited similar stability and activity characteristics compared to those modified with active N-hydroxysuccinimdyl esters. The activation of hydroxy- or amino-terminating compounds with DSC represents a general method that can be applied to any ligand which contains these functional groups for its covalent coupling to amines
Avidin derivatives and uses thereof
No full textA compound of the formula I: ##STR1## wherein R is H or --N.dbd.N-2-carboxyphenyl; A is (CH.sub.2).sub.n or --CH.dbd.CH--, wherein n is an integer from 0 to 10, or A may also be --CH(COOH)-- when R is --N.dbd.N-2-carboxyphenyl; and X is a radical selected from the group consisting of: (i) Cl; (ii) COOR.sub.1, wherein R.sub.1 is p-nitrophenyl or N-succinimidyl; (iii) CONH--NHR.sub.2, wherein R.sub.2 is H, COO(t-butyl) or COObenzyl; (iv) CONH--[B]--NHR.sub.3, wherein R.sub.3 is H, COOR.sub.1, or CO--[B']--maleimido, wherein R.sub.1 is t-butyl, p-nitrophenyl or N-succinimidyl, and B and B', the same or different, are (CH.sub.2).sub.n wherein n is an integer from 2 to 10; (v) CONH--[B]--COOR.sub.4, wherein R.sub.4 is H, C.sub.1 -C.sub.8 alkyl, N-succinimidyl; (vi) CONH--[B]--OH; (vii) CONH--[B]--CONH--NHR.sub.2, wherein R.sub.2 is H, COO(t-butyl) or COObenzyl; and (viii) NHR.sub.2, wherein R.sub.2 is H, COO(t-butyl) or COObenzyl, when A is --CH(COOH)-- and R is --N.dbd.N-2-carboxyphenyl. The 4'-hydroxyazobenzene-2-carboxylic acid (HABA) compounds are novel reagents for labeling, isolation and detection of biological molecules
DNA condensation by high-affinity interaction with avidin.
No full textAvidin, the basic biotin-binding glycoprotein from chicken egg white, is known to interact with DNA, whereas streptavidin, its neutral non-glycosylated bacterial analog, does not. In the present study we investigated the DNA-binding properties of avidin. Its affinity for DNA in the presence and absence of biotin was compared with that of other positively charged molecules, namely the protein lysozyme, the cationic polymers polylysine and polyarginine and an avidin derivative with higher isoelectric point (pI approximately 11) in which most of the lysine residues were converted to homoarginines. Gel-shift assays, transmission electron microscopy and dynamic light scattering experiments demonstrated an unexpectedly strong interaction between avidin and DNA. The most pronounced gel-shift retardation occurred with the avidin-biotin complex, followed by avidin alone and then guanidylated avidin. Furthermore, ultrastructural and light-scattering studies showed that avidin assembles on the DNA molecule in an organized manner. The assembly leads to the formation of nanoparticles that are about 50-100 nm in size (DNA approximately 5 kb) and have a rod-like or toroidal shape. In these particles the DNA is highly condensed and one avidin is bound to each 18 +/- 4 DNA base pairs. The complexes are very stable even at high dilution ([DNA] =10 pM) and are not disrupted in the presence of buffers or salt (up to 200 mM NaCl). The other positively charged molecules also condense DNA and form particles with a globular shape. However, in this case, these particles disassemble by dilution or in the presence of low salt concentration. The results indicate that the interaction of avidin with DNA may also occur under physiological conditions, further enhanced by the presence of biotin. This DNA-binding property of avidin may thus shed light on a potentially new physiological role for the protein in its natural environment
Azobenzene derivatives as labeling agents and intermediates thereof
No full textA compound of the formula I: ##STR1##wherein R is H or --N.dbd.N-2-carboxyphenyl; A is (CH.sub.2).sub.n or --CH.dbd.CH--, wherein n is an integer from 0 to 10, or A may also be --CH(COOH)-- when R is --N.dbd.N-2-carboxyphenyl; and X is a radical selected from the group consisting of: (i) Cl; (ii) COOR.sub.1, wherein R.sub.1 is p-nitrophenyl or N-succinimidyl; (iii) CONH--NHR.sub.2, wherein R.sub.2 is H, COO(t-butyl) or COObenzyl; (iv) CONH--[B]--NHR.sub.3, wherein R.sub.3 is H, COOR.sub.1, or CO--[B']--maleimido, wherein R.sub.1 is t-butyl, p-nitrophenyl or N-succinimidyl, and B and B', the same or different, are (CH.sub.2).sub.n wherein n is an integer from 2 to 10; (v) CONH--[B]--COOR.sub.4, wherein R.sub.4 is H, C.sub.1 -C.sub.8 alkyl, N-succinimidyl; (vi) CONH--[B]--OH; (vii) CONH--[B]--CONH--NHR.sub.2, wherein R.sub.2 is H, COO(t-butyl) or COObenzyl; and (viii) NHR.sub.2, wherein R.sub.2 is H, COO(t-butyl) or COObenzyl, when A is --CH(COOH)-- and R is --N.dbd.N-2-carboxyphenyl. The 4'-hydroxyazobenzene-2-carboxylic acid (HABA) compounds are novel reagents for labeling, isolation and detection of biological molecule
A chemical approach to illustrate the principle of signal transduction cascades using the avidin-biotin-system
No full textA new approach to illustrate the principle of signal transduction and to assemble protein multilayers is described. It is based on competing affinities of two different ligands for the same binding site of a protein.
A low-affinity ligand can be attached coValently to the protein, where it will be buried in the binding site and thus be prevented to interact with other proteins that recognize it. However, if a high-affinity ligand (or a molecule containing this ligand) is added, it will displace the low-affinity ligand (which still remains covalently
bound) from the binding site to the periphery. The low-affinity ligand is now available for interaction with other molecules, thus providing the means through which to assemble multilayers of proteins by a “recognition cascade”. This principle was demonstrated using the protein avidin which binds two ligands, biotin and 4-hydroxyazobenzene-2-carboxylic acid (HABA), with markedly different affinities. Avidin was affinity labeled with HABA, the low-affinity ligand, to produce a red, covalently conjugated avidin-HABA derivative (red avidin). Anti-HABA antibodies failed to recognize HABA buried in the binding site of avidin. However, upon addition of the high-affinity ligand biotin, HABA was expelled from the binding site and immediately
bound by the antibodies. Multilayer assemblies of HABAylated avidin and biotinylated anti-HABA antibodies could thus be constructed. This concept may find application in numerous fields, such as medicine, diagnostics, nanotechnology, and artificial intelligence
AVIDIN DERIVATIVES AND USES THEREOF
No full textA compound of formula (I) wherein R is H or -N=N-2-carboxyphenyl; A is (CH2)n or -CH=CH-, wherein n is an integer
from 0 to 10, or A may also be -CH(COOH)- when R is -N=N-2-carboxyphenyl; and X is a radical selected from the group
consisting of: (i) Cl; (ii) COOR1, wherein R1 is p-nitrophenyl or N-succinimidyl; (iii) CONH-NHR2, wherein R2 is H, COO(tbutyl)
or COObenzyl; (iv) CONH-[B]-NHR3, wherein R3 is H, COOR1, or CO-[B']-maleimido, wherein R1 is t-butyl, pnitrophenyl
or N-succinimidyl, and B and B', the same or different, are (CH2)n wherein n is an integer from 2 to 10; (v) CONH-
[B]- COOR4, wherein R4 is H, C1-C8 alkyl, N-succinimidyl; (vi) CONH-[B]-OH; (vii) CONH-[B]-CONH-NHR2, wherein R2
is H, COO(t-butyl) or COObenzyl; and (viii) NHR2, wherein R2 is H, COO(t-butyl) or COObenzyl, when A is -CH(COOH)- and
R is -N=N-2-carboxyphenyl. The 4'-hydroxyazobenzene-2-carboxylic acid (HABA) compounds are novel reagents for labeling, isolation and detection of biological molecules
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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