Abstract
New heterobifunctional cross-linking reagents that possessed a photoactive terminus, an electrophilic terminus, and a linking arm between the two termini that had a radiolabeled, enzymatically cleavable bond were synthesized. In a model study, succinimidyl N-[N′-(4-azidobenzoyl)tyrosyl]-β-alanate (16A) was coupled to n-butylamine (a Lys surrogate), iodinated, and cleaved with chymotrypsin in the presence of tyrosylamide to afford the desired adduct N-(N′-(4-azidobenzoyl)-3-iodotyrosyl)tyrosinamide, thereby demonstrating the feasibility of the enzymatic cleavage. In a biochemical study, succinimidyl N-[N′-(3-azido-5-nitrobenzoyl)tyrosyl]-β-alanate (16C) was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was successfully photo-cross-linked, in a calciumdependent manner, to the human erythrocyte plasma membrane Ca2+,Mg2+-ATPase and to a synthetic fragment (M13) containing the CaM-binding region of myosin light-chain kinase. In the latter case, densitometry readings indicated 20% cross-linking efficiency.
Original language | English |
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Pages (from-to) | 138-143 |
Number of pages | 6 |
Journal | Bioconjugate Chemistry |
Volume | 1 |
Issue number | 2 |
DOIs | |
State | Published - Mar 1 1990 |
Bibliographical note
Funding Information:We thank the NationalScience Foundation (Grant CHE- 8607441) fortheir financial support, K. Drauz andA. Bernd ofDegussaAG for agenerous gift ofamino acids, NATO for a travel grant (Grant RG #0346/88) to D.S.W. and A.D.,andtheUniversityofKentuckyforthepurchase ofbond issueequipmentandforFacultyGrantInitia- tiveAwards (toD.S.W.andT.C.V.).
ASJC Scopus subject areas
- Biotechnology
- Bioengineering
- Biomedical Engineering
- Pharmacology
- Pharmaceutical Science
- Organic Chemistry