Lanthanide probes in biological systems: The calcium binding site of pancreatic elastase as studied by terbium luminescence

Bruce Martin R. (1980) Lanthanide probes in biological systems: The calcium binding site of pancreatic elastase as studied by terbium luminescence. Biochemistry, 19 6: 1127-1132.

Author Bruce Martin R.
Title Lanthanide probes in biological systems: The calcium binding site of pancreatic elastase as studied by terbium luminescence
Journal name Biochemistry   Check publisher's open access policy
ISSN 0006-2960
Publication date 1980-01-01
Sub-type Article (original research)
Open Access Status Not yet assessed
Volume 19
Issue 6
Start page 1127
End page 1132
Total pages 6
Subject 1303 Specialist Studies in Education
Abstract In the presence of porcine pancreatic elastase, Tb3+ ions can be excited by energy transfer from an aromatic side chain of the protein. The enhancement of Tb3+ luminescence (λex = 280 nm; λem = 543 nm) due to binding of Tb3+ to elastase is ∼2 × 104. The luminescence excitation spectrum indicates that energy transfer is from a tryptophan side chain to the bound Tb3+. Analysis of the luminescence intensity data shows that Tb3+ ions bind to elastase, forming a 1:1 complex, with an association constant of 3 × 105 M-1 (pH 6.58, 25 °C). Binding constants for Ca2+, Mg2+, and Zn2+ to elastase were also determined by the luminescence method (displacement of Tb3+), showing that under physiological conditions elastase exists as Ca2+·elastase. Addition of the competitive inhibitor acetyltrialanine to Tb3+-elastase causes a twofold enhancement of luminescence intensity. By measuring the effect of acetyltrialanine concentration on the intensity, it was shown that this effect is due to binding of the inhibitor to the Tb3+-enzyme, and a dissociation constant Kd was determined. Kd was found to be a factor of 4 smaller than Ki for acetyltrialanine as a competitive inhibitor of the hydrolysis of succinyltrialanine p-nitroanilide, determined under the same conditions. This and other evidence indicates that acetyltrialanine can bind to two distinct sites in Tb3+-elastase. This finding requires reassessment of other work using lanthanide probes of calcium binding sites in elastase and other enzymes.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: Scopus Import - Archived
 
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Created: Sat, 09 Jul 2016, 15:42:24 EST by System User