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

Abstract

1. 4-(N-2-Aminoethyl2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole (compound I) was synthesized and evaluated as a fluorescent labelling reagent for thiol groups. 2. The design of compound (I) as one example of a general type of reporter group delivery reagent (2-pyridyl-S-S-X, where X contains an environmentally sensitive spectroscopic probe) is discussed. 3. The electronic absorption spectrum of compound (I) was determined over a wide range of pH and the spectral changes that accompany its reaction with low-molecular-weight thiols, e.g. L-cysteine, and with papain (EC 3.4.22.2) and bovine serum albumin are discussed. 4. A new value of epsilon343 for 2-thiopyridone (Py-2-SH) was determined as 8.08 X 10(3) +/- 0.08 X 10(3)M-1-cm-1. 5. Spectral analysis of the reactions of compound (I) with L-cysteine and with papain (in the pH range 3.5-8.0) showed that even under equimolar conditions the reaction (thiol-disulphide interchange to release Py-2-SH) is essentially stoicheimoetric and probably proceeds by specific attack at the sulphur atom distal from the pyridyl ring of compound (I). 6. The fluorescence-emission spectra of compound (I) and of the products of its reaction with papain and with ficin (EC 3.4.22.3) were determined. Compound (I) is highly fluorescent in aqueous solution. Excitation within the intense visible absorption band (lambda max. 481 nm, epsilon max. 2.52 X 10(4)M-1-cm-1) provides green fluorescence with an emission maximum at 540 nm. Both papain and ficin labelled by reaction with compound (I) are characterized by fluorescence-emission maxima (535 nm and 530 nm respectively) of even higher intensity. The fluorescence emission of the product of the reaction of papain with compound (I) was shown to be 25 times more intense than that of the product of the reaction of papain with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride). 7. The second-order rate constants (k2) for the reactions of compound (I) and of Nbd chloride with GSH, papain, albumin, ficin, 2-benzimidazolylmethanethiol and 2-benzimidazolylethanethiol were determined at 25.0 degrees C and various pH values. At pH4 the values of k2(compound I)/k2(Nbd chloride) are: GSH, 288; albumin, 36; papain 3 X 10(3); ficin, 3 X 10(4). 8. The pH-k2 profiles for the reactions of compound (I) and of Nbd chloride with the two 2-benzimidazolylalkanethiols were determined. Of the four profiles only that for the reaction of compound (I) with 2-benzimidazolylmethanethiol is characterized by a striking rate maximum in acidic media.

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

“…[H+])}M-1 cm- (Little, 1971;Brocklehurst & Little, 1973;Stuchbury et al, 1975). In reactions with Nbs2 and N-ethylmaleimide, concentrations were calculated by using AS412= 1.36 x 104M1 -cm-' (Ellman, 1959) and A310 --620M-1 *cm1 (Andrews & Reithel, 1970) (ii) A given solution of urease (2ml of 1-2aMM protoin) was titrated in a stepwise procedure with 10Q1 portions of 1.5mM-2-Py-S-S.-2-Py.…”

Coupling of protein antigens to erythrocytes through disulfide bond formation: preparation of stable and sensitive target cells for immune hemolysis.

“…Briefly, one ml of a properly diluted PDTP-Ag solution (0.5-1.0 mg/ml) in phosphate-buffered saline was treated with 0.1 ml of 1 M dithiothreitol at room temperature for 40 min. This treatment effects the release of pyridine-2-thione, which has a molar absorptivity of8.08 x 103 at 343 nm (19). The amount of pyridine-2-thione released is equivalent to the content of PDTP groups in PDTP-Ag.…”

Targeting of Porcine Pancreatic Phospholipase A2 to Human Platelets

“…The reactions were followed by measuring the release of pyridine-2-thione at 343 nm using a specific absorption of 8.08 mM-' . cm-' [21]. After coupling of the peptide, the PLA derivatives were dialysed for 60 h against 0.1 % acetic acid at 4°C to remove the unreacted peptide and pyridine-2-thione.…”

“…PLA is secreted into the gut as a precursor enzyme which lacks a free a-amino group, since the N-terminal amino acid is S-oxoproline (pyroglutamic acid) [I 21. Thus, when the precursor is used for acylation of amino groups, only the t-amino groups of the lysine residues are accessible.…”