Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent. (original) (raw)

• Journal List
• Biochem J
• v.173(3); 1978 Sep 1
• PMC1185838

Biochem J. 1978 Sep 1; 173(3): 723–737.

Abstract

A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves two steps. First, 2-pyridyl disulphide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester sie of the reagent. The protein-bound 2-pyridyl disulphide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulphide bonds. The technique has been used for the introduction of thiol groups de novo into ribonuclease, gamma-globulin, alpha-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulphide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, for example, the method described above) via thiol-disulphide exchange to form disulphide-linked protein-protein conjugates. This conjugation technique has been used for the preparation of an alpha-amylase-urease, a ribonuclease-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulphide bridges between the protein molecules can easily be split by reduction or by thiol-disulphide exchange. Thus conjugation is reversible. This has been demonstrated by scission of the ribonuclease-albumin and the alpha-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate has been prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23 degrees C).

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• Axén R, Myrin PA, Janson JC. Chemical fixation of chymotrypsin to water-insoluble crosslinked dextran (sephadex) and solubilization of the enzyme derivatives by means of dextranase. Biopolymers. 1970;9(4):401–413. [PubMed] [Google Scholar]
• Axén R, Carlsson J, Janson JC, Porath J. Ribonuclease chemically attached to beads of epichlorohydrin cross-linked agarose. Enzymologia. 1971 Dec 31;41(6):359–364. [PubMed] [Google Scholar]
• Blakeley RL, Webb EC, Zerner B. Jack bean urease (EC 3.5.1.5). A new purification and reliable rate assay. Biochemistry. 1969 May;8(5):1984–1990. [PubMed] [Google Scholar]
• Brocklehurst K, Little G. Reactivities of the various protonic states in the reactions of papain and of L-cysteine with 2,2'- and with 4,4'- dipyridyl disulphide: evidence for nucleophilic reactivity in the un-ionized thiol group of the cysteine-25 residue of papain occasioned by its interaction with the histidine-159-asparagine-175 hydrogen-bonded system. Biochem J. 1972 Jun;128(2):471–474. [PMC free article] [PubMed] [Google Scholar]
• Brocklehurst K, Carlsson J, Kierstan MP, Crook EM. Covalent chromatography. Preparation of fully active papain from dried papaya latex. Biochem J. 1973 Jul;133(3):573–584. [PMC free article] [PubMed] [Google Scholar]
• Browne DT, Kent SB. Formation of non-amidine products in the chemical modification of horse liver alcohol dehydrogenase with imido esters. Biochem Biophys Res Commun. 1975 Nov 3;67(1):133–138. [PubMed] [Google Scholar]
• Carlsson J, Svenson A. Preparation of bovine mercaptalbumin by means of covalent chromatography. FEBS Lett. 1974 Jun 1;42(2):183–186. [PubMed] [Google Scholar]
• Carlsson J, Gabel D, Axén R. Competitive inhibition of Sephadex-bound chymotrypsin and trypsin. Hoppe Seylers Z Physiol Chem. 1972 Dec;353(12):1850–1854. [PubMed] [Google Scholar]
• Carlsson J, Axén R, Brocklehurst K, Crook EM. Immobilization of urease by thiol-disulphide interchange with concomitant purification. Eur J Biochem. 1974 May 2;44(1):189–194. [PubMed] [Google Scholar]
• Carlsson J, Axén R, Unge T. Reversible, covalent immobilization of enzymes by thiol-disulphide interchange. Eur J Biochem. 1975 Nov 15;59(2):567–572. [PubMed] [Google Scholar]
• COHEN S, PORTER RB. STRUCTURE AND BIOLOGICAL ACTIVITY OF IMMUNOGLOBULINS. Adv Immunol. 1964;27:287–349. [PubMed] [Google Scholar]
• Cuatrecasas P, Parikh I. Adsorbents for affinity chromatography. Use of N-hydroxysuccinimide esters of agarose. Biochemistry. 1972 Jun 6;11(12):2291–2299. [PubMed] [Google Scholar]
• Egorov TA, Svenson A, Rydén L, Carlsson J. A rapid and specific method for isolation of thiol-containing peptides from large proteins by thiol-disulfide exchange on a solid support. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3029–3033. [PMC free article] [PubMed] [Google Scholar]
• Grassetti DR, Murray JF., Jr Determination of sulfhydryl groups with 2,2'- or 4,4'-dithiodipyridine. Arch Biochem Biophys. 1967 Mar;119(1):41–49. [PubMed] [Google Scholar]
• JUNGE JM, STEIN EA, NEURATH H, FISCHER EH. The amino acid composition of alpha-amylase from Bacillus subtilis. J Biol Chem. 1959 Mar;234(3):556–561. [PubMed] [Google Scholar]
• KLAPPER MH, HACKETT DP. INVESTIGATIONS ON THE MULTIPLE COMPONENTS OF COMMERCIAL HORSERADISH PEROXIDASE. Biochim Biophys Acta. 1965 Feb 22;96:272–282. [PubMed] [Google Scholar]
• LINDLEY H. A study of the kinetics of the reaction between thiol compounds and choloracetamide. Biochem J. 1960 Mar;74:577–584. [PMC free article] [PubMed] [Google Scholar]
• Marshall JJ. Preparation and characterization of a dextran-amylase conjugate. Carbohydr Res. 1976 Jul;49:389–398. [PubMed] [Google Scholar]
• Mattiasson B, Mosbach K. Studies on a matrix-bound three-enzyme system. Biochim Biophys Acta. 1971 Apr 14;235(1):253–257. [PubMed] [Google Scholar]
• Mosbach K, Mattiasson B. Matrix-bound enzymes. II. Studies on a matrix-bound two-enzyme-system. Acta Chem Scand. 1970;24(6):2093–2100. [PubMed] [Google Scholar]
• Perham RN, Thomas JO. Reaction of tobacco mosaic virus with a thiol-containing imidoester and a possible application to X-ray diffraction analysis. J Mol Biol. 1971 Dec 14;62(2):415–418. [PubMed] [Google Scholar]
• Stuchbury T, Shipton M, Norris R, Malthouse JP, Brocklehurst K, Herbert JA, Suschitzky H. A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety. Biochem J. 1975 Nov;151(2):417–432. [PMC free article] [PubMed] [Google Scholar]

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