Summary
The two isothiocyanate groups of the anion transport inhibitor 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonate (H2DIDS) may react covalently with two lysine residues calleda andb that reside on the chymotryptic 60,000 Dalton and 35,000 Dalton segments, respectively, of the band 3 protein of the human erythrocyte membrane. Under suitable conditions, the reaction leads to the establishment of intramolecular cross-links betweena andb (M.L. Jennings & H. Passow, 1979,Biochim. Biophys. Acta 554:498–519). In the present work, the time course of the reactions witha andb, and of the establishment of the cross-link were investigated experimentally and compared with simple mathematical models of the reaction sequence. The rates of reaction witha andb were found to increase with increasing pH. Regardless of pH, the rate of reaction witha exceeds that withb several-fold. One the H2DIDS molecule has reacted witha, the rate of the subsequent reaction of the other isothiocyanate group withb is reduced by about 1/30. The reactions that follow the unilateral, attachment to siteb are not yet clear. A more detailed analysis of the time course of the cross-linking reaction suggests that a satisfactory description of the kinetics requires the assumption that the H2DIDS binding site may exist in two different states, and that the transition from one state to the other is associated with changes of the reactivities, of either lysa alone or of both lysa andb. This led to the formulation of the two-states model of the H2DIDS binding site, which is supported by other pieces of independent evidence. The analysis of the pH dependence of the rate of thiocyanylation ofb shows that the apparent pK value of that lysine residue is about 9.9 to 10.0 and hence slightly lower than the intrinsic pK of a lysine residue in an aqueous environment.
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Abbreviations
- APMB:
-
2-(4-amino-3-sulfophenyl)-6-methyl-7-benzothia-zolsulfonic acid
- DANS-Cl:
-
5-(dimethylamino)naphthalenesulfonyl chloride
- DAS:
-
diacetamido stilbene-2,2′-disulfonate
- DBDS:
-
4,4′-dibenzoyl stilbene-2,2′-disulfonate
- DNDS:
-
4,4′-dinitro stilbene-2,2′-disulfonate
- EDTA:
-
ethylenediaminetetraacetic acid
- H2DIDS:
-
4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid
- N2ph-F:
-
1-fluoro-2,4-dinitrobenzene
- PCMBS:
-
para-chloromercuribenzoate
- SDS:
-
sodium dodecyl sulfate
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Kampmann, L., Lepke, S., Fasold, H. et al. The kinetics of intramolecular cross-linking of the band 3 protein in the red blood cell membrane by 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid (H2DIDS). J. Membrain Biol. 70, 199–216 (1982). https://doi.org/10.1007/BF01870563
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DOI: https://doi.org/10.1007/BF01870563