The accessibility of protein-bound dinitrophenyl groups to univalent fragments of anti-dinitrophenyl antibody

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1. The kinetic parameters Kcat. and Km were determined for the hydrolysis of some arginine naphthylamides by human cathepsin B. 2. A new and efficient synthesis of Z-Arg-Arg-NNap (benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide) was developed. 3. Z-Arg-Arg-NNap was a specific and sensitive substrate for cathepsin B, and was used for kinetic studies. 4. Values of kcat. were maximal in the pH range 5.4--6.2, and depended on a single ionizing group of pKa 4.4. 5. Leupeptin was a purely competitive inhibitor of human cathepsin B. 6. The effect of pH on the apparent inhibitor constant, Ki (app.), was determined. Ki (app.) was pH-independent in the range pH 4.3--6.0, with the mean value 7 x 10(-9) M.

Section: Methodsmentioning

confidence: 99%

Human cathepsin B. Application of the substrate N-benzyloxycarbonyl-l-arginyl-l-arginine 2-naphthylamide to a study of the inhibition by leupeptin

Knight¹

1980

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“…Anti-Dnp antibody was produced in rabbits (Eisen, 1964) and purified as described by Eisen et al (1967). A univalent (Fab) fragment preparation (Knight & Green, 1976) gave only one protein band on slab-gel electrophoresis (Davies et al, 1978), corresponding to mol.wt. 45000.…”

Section: A Nti-dnp Antibodymentioning

confidence: 99%

“…Values of the apparent dissociation constant, Kapp.' were calculated as described by Knight & Green (1976 0.1 x 10-9M at 0.04,uM-antibody, indicating that most of the antibody species in the preparation bound the hapten with Kapp < 10-10M (cf. Knight & Green, 1976).…”

Section: A 280omentioning

confidence: 99%

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Interaction of dinitrophenyl-pepstatins with human cathepsin D and with anti-dinitrophenyl antibody. Development of potential reagents for the localization in vivo of active proteinases at sites of tissue injury

Knight¹,

Hornebeck²,

Matthews³

et al. 1980

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Extracellular cathepsin D has been observed by various cytochemical methods at sites of tissue injury. However, the role of this enzyme in connective tissue matrix degradation is uncertain because there are no histochemical methods for determining whether or not the cathepsin D is active at such sites in living tissues. We considered that the combined use of a labelled tight-binding inhibitor with immunoprecipitation of the enzymes might overcome this problem. We have explored the application of derivatives of the inhibitor pepstatin, as only active cathepsin D binds pepstatin tightly. A series of N-pepstatinyl-N'-dinitrophenyl-alpha, omega-diaminoalkanes were synthesized with alkyl-chain lengths of two, four and six carbon atoms. These compounds were tight-binding inhibitors of human cathepsin D. In fluorescence-quenching titrations the dinitrophenyl groups were also fully available to bind high-affinity anti-dinitrophenyl antibody. It was shown by immunodiffusion in gels and by gel permeation chromatography that N-pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane was a bifunction inhibitor able to bind cathepsin D and anti-dinitrophenyl antibody at the same time.

“…We have described previously (Knight & Green, 1976) a new technique for probing the environment of the amino acid residues in proteins that can be labelled with Dnp groups. Fluoresence-quenching titrations showed that Dnp groups attached by aminoalkylsuccinimide chains to the thiol group of bovine mercaptalbumin were fully available to univalent fragments of high-affinity antibody to Dnp, even when the alkyl chain was short.…”

mentioning

confidence: 99%

Interaction of dinitrophenyl groups bound to bovine serum albumin with univalent fragments of anti-dinitrophenyl antibody

Knight¹,

Green²

1979

Two lysine residues of bovine serum albumin reacted with 1-fluoro-2,4-dinitrobenzene with apparent second-order rate constants approx. 500-times greater than those observed in similar reactions with low-molecular-weight lysine derivatives. A series of dinitrophenyl (Dnp)-bovine serum albumins were prepared and their ability to bind univalent fragments of anti-Dnp antibody was measured by fluorescence-quenching titrations. Compared with the Dnp group of the free hapten, 6-N-Dnp-aminohexanoate, the majority of the protein-bound Dnp groups were unavailable to the antibody at pH8.0. When the same Dnp-albumins were titrated at pH3.0 the availability of the Dnp groups increased approx. 3-fold. Dnp-albumins were treated with pepsin at pH3.0 and Dnp-containing fragments isolated by chromatography on DE-52 DEAE-cellulose. Fluorescence-quenching titrations showed that the Dnp groups on the fragments behaved like the free hapten with respect to quenching efficiency, although with an increased dissociation constant. The association between the Dnp-albumins and the antibody was measured also by difference-spectral titrations at high protein concentrations. Antibody binding was increased under these conditions, but the Dnp group of mono-Dnp-albumin remained unavailable to antibody. We propose that the reactive lysine residues are located in clefts between the globular sub-domains of the single polypeptide chain. Dnp groups attached to these lysine residues are fully exposed to the solvent, but binding of the macromolecular probe, anti-Dnp antibody, is sterically hindered by the adjacent surface of the albumin molecule.