Pyridyldithio Propionate

Antibody containing, nu,if1v rfithin PDTP-activated antibody 

Since activated molecules and crosslinks formed between two species are identical to those formed using SPDP, it is of little advantage to use PDTP. Furthermore, an EDC-mediated reaction of the carboxylate end of the crosslinker with amine groups on proteins can cause concomitant zero-length crosslinking and polymerization of protein molecules. For these reasons, SPDP is the better choice for preparing conjugates. 

A final method of forming disulfide crosslinks between toxins and targeting molecules is the use of S-sulfonate formation using sodium sulfite (Na2SC 3) in the presence of sodium tetrathion-ate (Na2S40g). Tetrathionate reacts with sulfhydryls to form sulfenylthiosulfate intermediates (section 1.1.5.2). These derivatives are reactive toward other thiols to create disulfide linkages 

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SPDP, N-succinimidyl-3-(2-pyridyldithio)propionate, is one of the most popular heterobifunctional crosslinking agents available. The activated NHS ester end of SPDP reacts with amine... 

N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) also may be used to activate pure PE lipids in a similar manner to SMPB. The result will be a derivative containing pyridyl disulfide groups rather than maleimide groups (Figure 22.12). Pyridyl disulfides react with sulfhydryls to form disulfide linkages. Either the standard SPDP or the long-chain version, LC-SPDP, may be employed in the following protocol. 

Conjugates of (strept)avidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to create a sulf-hydryl-reactive derivative, followed by modification of (strept)avidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of (strept)avidin can be found in Section 3.1, this chapter. The procedure for SPDP activation of phycobiliproteins can be found in Chapter 9, Section 7. Reacting the SPDP-activated phycobiliprotein with thiol-labeled (strept)avidin at a molar ratio of 2 1 will result in highly fluorescent biotin binding probes. 

In a somewhat similar scheme, Noguchi et al. (1992) prepared a carboxylate spacer arm by reacting 6-bromohexanoic acid with a dextran polymer. The carboxylate then was aminated with ethylene diamine to form an amine-terminal spacer (Figure 25.15). This dextran derivative finally was reacted with N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Chapter 5, Section 1.1) to create the desired sulfhydryl-reactive polymer (Section 2.4, this chapter). The SPDP-activated polymer then could be used to prepare an immunoconjugate composed of an antibody against human colon cancer conjugated with the drug mitomycin-C. 

Carlsson, J., Drevin, H., and Axen, R. (1978) Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3(2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem. J. 173, 723-737. 

Singh, V., Mavila, A.K., and Kar, S.K. (1993) Comparison of the cytotoxic effect of hormonotoxins prepared with the use of heterobifunctional cross-linking agents N-succinimidyl 3-(2-pyridyldithio)propionate and N-succinimidyl 6-[3-(2-pyridyldithio)propionamido]-hexanoate. Bioconjugate Chem. 4, 473M82. 

CaM was purified from porcine brain. The purity of proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. CaM and PDE were cross-linked with l-ethyl-3(3-dimethylamino-propyl) carbodiimide (EDC) or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) in a buffer solution of 0.1 M HEPES (pH 7.1) in the presence of 1 mM CaCl2. After a buffer solution containing 2 mM EGTA added into the reaction solution, the CaM-PDE hybrid was separated from other ingredients by gel chromatography on a Sepharose CL-6B solumn. 

Many types of mono-, bi-, and multifunctional coupling reagents are available for labeling antibodies or antigens with an enzyme. Glutaraldehyde, carbodiimide, N-succinimidyl-3-(2-pyridyldithio)propionate, and periodate oxidation of carbohydrate moieties to form active dialdehydes are several commonly used approaches in the preparation of enzyme conjugates (104-106). 

N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) is by far the most popular heterobifunctional cross-linking agent used for immunotoxin conjugation (Chapter 5, Section 1.1). The activated NHS ester end of SPDP reacts with amine groups in one of the two proteins to form an amide linkage. The 2-pyridyldithiol group at the other end... 

Ansell, S. M.,Tardi, P. G., and Buchkowsky, S. S. (1996), 3-(2-Pyridyldithio)propionic acid hydrazide as a cross-linker in the formation of liposome-antibody conjugates, Bioconjugate Chem.,1,490-496. 

Sulfophenylazo)-l,8-dihydroxy-3,6-naphthalenedisulfonic acid (trisodium salt) A-Succinimidyl 3-(2-pyridyldithio)propionate... 

Cross-linking compound ]3-(2-pyridyldithio) propionic acid iV-hydroxy-succinimide ester (SPDP) (Sigma, Poole, UK). 

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