Metabolism of 25-hydroxycholecalciferol in human promyelocytic leukemia cells (HL-60). Isolation and identification of (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol

Human promyelocytic leukemia cells incubated with 25-hydroxy[26,27-methyl-3H] cholecalciferol (1 microCi) or non-radioactive 25-hydroxycholecalciferol (550 micrograms) produced significant quantities of two vitamin D3 metabolites. The two metabolites were isolated and purified by methanol chloroform extraction and a series of chromatographic procedures. The metabolite purification and elution positions on these columns were followed by radioactivity and their ultraviolet absorption at 310 nm. The two metabolites have been unequivocally identified as (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol by ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry and co-chromatography with synthetic compounds on a high-performance liquid chromatograph. (5E)- but not (5Z)-19-nor-10-oxo-25-hydroxycholecalciferol was able to induce HL-60 cell phenotypic and functional differentiation. However, these two metabolites of 25-hydroxycholecalciferol did not bind specifically to the chick intestinal 3.7 S. receptor protein for 1 alpha,25-dihydroxycholecalciferol. The precise biological role of these metabolites is as yet unclear.     

Human myeloid leukemia cells metabolize 25-hydroxyvitamin D3 in vitro

Human promyelocytic leukemia cells (HL-60) converted 25-hydroxyvitamin D3 to two more polar metabolites during in vitro incubations. A two-step high pressure liquid chromatography system revealed two unique elution positions of those leukemic cell-derived metabolites that exactly co-migrated with the elution positions of 5(Z)-19-nor-10-oxo-25-hydroxyvitamin D3 and 5(E)-19-nor-10-oxo-25-hydroxyvitamin D3, respectively. These unique metabolites did not bind specifically to a protein receptor for 1,25-dihydroxyvitamin D3.     

Phagocytic cells metabolize 25-hydroxyvitamin D3 to 10-oxo-19-nor-25-hydroxyvitamin D3 and a new metabolite, 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one

The metabolism of 25-hydroxyvitamin D3 [25(OH)D3] was examined in several phagocytic cells including alveolar macrophages and myeloid leukemia cells (M1, HL-60 and U937). Phagocytic cells converted 25(OH)D3 to 10-oxo-19-nor-25-hydroxyvitamin D3 and a new metabolite. The former metabolite was dominant in shorter incubation periods (1 h), whereas the latter dominated over longer incubation periods (24 h). The new metabolite was produced from 25(OH)D3 directly but not through 10-oxo-19-nor-25-hydroxyvitamin D3. The new metabolite was unequivocally identified as 8 alpha,25-dihydroxy-9-10-seco-4,6,10(19)-cholestatrien-3-one. These results suggest that phagocytic cells somehow promote oxidation of the triene part of vitamin D compounds.     

Isolation, identification, and chemical synthesis of 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one. A new metabolite of 25-hydroxyvitamin D3 produced by mouse myeloid leukemia cells (M1)

It is known that phagocytic cells such as monocyte-macrophages and myeloid leukemia cells metabolize 25-hydroxyvitamin D3 to 10-oxo-19-nor-25-hydroxyvitamin D3. Now we have found that phagocytic cells metabolize 25-hydroxyvitamin D3 not only to 10-oxo-19-nor-25-hydroxyvitamin D3 but also to a new metabolite eluted just after 24R,25-dihydroxyvitamin D3 on straight phase high pressure liquid chromatography with a 2-propanol-hexane solvent system. The new metabolite, produced by murine myeloid leukemia cells (M1), was isolated in pure form and identified as 8,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one on the basis of mass, ultraviolet, infrared, and proton magnetic resonance spectra. The 8 alpha-hydroxy epimer of the putative metabolite was chemically synthesized in two steps starting from 25-hydroxyvitamin D3. The spectral data and chromatographic behavior of chemically synthesized 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one coincided exactly with those of the isolated metabolite, indicating that the stereochemistry of the hydroxyl group at the 8-position is alpha. On the basis of the structural characteristics of the two metabolites produced from 25-hydroxyvitamin D3 (the present metabolite and 10-oxo-19-nor-25-hydroxyvitamin D3), it is suggested that dioxygenases are involved in the production of these metabolites from 25-hydroxyvitamin D3 in phagocytic cells.     

Occurrence of a novel nucleotide, Zpp5'A2'p, in rat liver extracts

The nucleotide Zpp5'A2'p has been isolated from rat liver. Z stands for an unknown compound, probably a nucleoside. The preliminary structure of Zpp5'A2'p has been elucidated by treatment with phosphodiesterase and/or alkaline phosphatase and analysis of the products of the reaction by high pressure liquid chromatography. The following ultraviolet absorption spectral characteristics were determined at pH 7.0: Zpp5'A2'p (lambda max = 265 nm; A250/A260 = 0.76; A280/A260 = 0.83); Zp (lambda max = 280 nm; A250/A260 = 0.88; A280/A260 = 1.46). The molar extinction coefficient found for Zp, at 280 nm, was (7.5 + 0.9) X 10(3) M-1 cm-1. The base of Zp could correspond to an indole derivative.     

Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.     

Isolation and identification of 1 alpha,25-dihydroxy-24-oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3: in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3

Three new in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3 were isolated from the serum of dogs given large doses (two doses of 1.5 mg/dog) of 1 alpha,25-dihydroxyvitamin D3. The metabolites were isolated and purified by methanol-chloroform extraction and a series of chromatographic procedures. By cochromatography on a high-performance liquid chromatograph, ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry, and specific chemical reactions, the metabolites were identified as 1 alpha,25-dihydroxy-24- oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3. According to these procedures, the total amounts of the isolated metabolites were as follows: 1 alpha,25-dihydroxyvitamin D3, 23.6 micrograms; 1 alpha,25-dihydroxy-24- oxovitamin D3, 1.8 micrograms; 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 9.2 micrograms; 1 alpha,24(R),25-trihydroxyvitamin D3, 15.4 micrograms; 1 alpha,24(S),25-trihydroxyvitamin D3, 1.0 microgram. With recovery corrections, the serum levels of each metabolite were approximately 49 ng/mL for 1 alpha,25-dihydroxyvitamin D3, 3.7 ng/mL for 1 alpha,25-dihydroxy-24- oxovitamin D3, 19 ng/mL for 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 32 ng/mL for 1 alpha,24(R),25-trihydroxyvitamin D3, and 2.1 ng/mL for 1 alpha,24(S),25-trihydroxyvitamin D3.     

Sarcoid granulomas metabolize 25-hydroxyvitamin D3invitro

Sarcoid granulomas metabolized 25-hydroxyvitamin D₃ to two unidentified metabolites during $\text{in}\text{vitro}$ incubation. A two-step high pressure liquid chromatography system revealed two unique elution positions of these sarcoid-derived metabolites that exactly comigrated with the elution positions of 5(Z)-19-nor-10-oxo-25(OH)D₃ and 5(E)-19-nor-10-oxo-25(OH)D₃, respectively. These unique metabolites did not bind specifically to a protein receptor for 1,25(OH)₂D₃.     

In vitro metabolism of 19-nor-1alpha, 25-(OH)2D2 in cultured cell lines: inducible synthesis of lipid- and water-soluble metabolites

The active vitamin D analog, 19-nor-1alpha,25-dihydroxyvitamin D2 (19-nor-1alpha,25-(OH)2D2), has a similar structure to the natural vitamin D hormone, 1a,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), but lacks the C10-19 methylene group and possesses an ergosterol/ vitamin D2 rather than a cholesterol/vitamin D3 side chain. We have used this analog to investigate whether any of these structural features has any effect upon the type and rate of in vitro metabolism observed. Using a vitamin D-target cell, the human keratinocyte, HPK1A-ras, we observed formation of a number of metabolites, three of which were purified by extensive HPLC and conclusively identified by a combination of GC-MS and chemical derivatization as 19-nor-1alpha,24,25-(OH) 3D2, 19-nor-1alpha,24,25,26-(OH) 4D2, and 19-nor-1alpha,24,25,28-(OH)4,D2. The first metabolite is probably a product of the vitamin D-inducible cytochrome P450, P450cc24 (CYP24), while the latter two metabolites are likely to be further metabolic products of 19-nor-1alpha,24,25-(OH)3D2. These hydroxylated metabolites resemble those identified by other workers as products of the metabolism of 1alpha,25-(OH)2D2 in the perfused rat kidney. It therefore appears from the similar metabolic fate of 19-nor-1alpha,25-(OH)2D2 and 1alpha,25-(OH)2D2 that the lack of the C10-19 methylene group has little effect upon the nature of the lipid-soluble metabolic products and the rate of formation of these products seems to be comparable to that of products of 1alpha,25-(OH)2D3 in vitamin D-target cells. We also found extensive metabolism of 19-nor-1alpha,25(OH)2D2 to water-soluble metabolites in HPK1A-ras, metabolites which remain unidentified at this time. When we incubated 19-nor-1alpha,25-(OH)2D2 with the liver cell line HepG2, we obtained only 19-nor-1alpha,24,25-(OH)3D2. We conclude that 19-nor-1alpha,25-(OH)2D2 is efficiently metabolized by both vitamin D-target cells and liver cells.     

Three new triterpenoids from Potentilla multicaulis

Three new triterpenoids, 19-hydroxy-2,3-secours-12-ene-2,3,28-trioic acid 3- methyl ester (1), 19-hydroxy-1-oxo-2-nor-2,3-secours-12-ene-3,28-dioic acid (2), and (3beta,18alpha,19alpha)-3,28-dihydroxy-20,28-epoxyursan-24-oic acid (3), were isolated from the roots of Potentilla multicaulis. Their structures were elucidated on the basis of spectroscopic methods (IR, HR-ESI-MS, and 1D- and 2D-NMR). Compound 2b exhibited moderate cytotoxic activity against human promyelocytic leukemia (HL-60) cells.     

Biotransformation of 20(S)-protopanaxatriol by Mucor spinosus and the cytotoxic structure activity relationships of the transformed products

Biotransformation of 20(S)-protopanaxatriol (1) by the fungus Mucor spinosus AS 3.3450 gave 10 metabolites (2-10), of which 2-5 were previously known. On the basis of NMR and MS analyses, structures 6-10 were established as 12-oxo-23beta-hydroxyl-20(S)-protopanaxatriol (6), 20S,24R-epoxy-dammaran-3beta,6alpha,25-triol-12-one (7), 29-hydroxyl-20(S)-protopanaxatriol (8a), 12-oxo-11beta-hydroxyl-20(S)-protopanaxatriol (8b), 28-hydroxyl-20(S)-protopanaxatriol (9) and 12-oxo-20(S)-protopanaxatriol (10). The biotransformation kinetics of 1 has been investigated and a possible biotransformation pathway proposed. The in vitro cytotoxic activities of metabolites against three human cancer cell lines were determined by the MTT method; compounds 8a, 9 and 10 had more potent inhibitory effects against HL-60 cell line than the substrate.     

C26-analogs of naturally occurring bile alcohols-II. Preparation of 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23-tetrols (23R and 23S) and 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrols (25R and 25S) by a hydroboration procedure

A convenient procedure for the synthesis of 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23-tetrol (23R and 23S) and 24-nor-5 beta-cholestane-3 alpha,7 alpha,12alpha,26-tetrol (25R and 25S) starting from 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol was developed. Dehydration of 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha, 25-tetrol with glacial acetic acid and acetic anhydride yielded a mixture of 24-nor-5 beta-cholest-23-ene-3 alpha,7 alpha,12 alpha-triol and the corresponding delta 25 compound. Hydroboration and oxidation of the mixture of unsaturated nor-triols resulted in the formation of 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23-tetrols (23R and 23S) and 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrols (25R and 25S). In addition, smaller amounts of 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,22 xi-tetrol and 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol were also obtained. The C26 bile alcohols epimeric at C-23 and C-25 were resolved by analytical and preparative TLC and characterized by gas-liquid chromatography and mass spectrometry. Provisional assignment of the configurations of the C-23 and C-25 hydroxyl groups were made on the basis of molecular rotation differences. These C26 alcohols will be used to test the stereospecificity of the hepatic enzymes that promote oxidation of the cholesterol side chain.     

Structural and biosynthetic studies of a principal bile alcohol, 27-nor-5beta-cholestane-3alpha,7alpha,12alpha,24,25-pentol, in human urine

The stereochemistry at C-24 and C-25 of 27-nor-5beta-cholestane-3alpha,7alpha,12alpha,24 ,25-pentol, a principal bile alcohol in human urine, and its biosynthesis are studied. Four stereoisomers of the C(26)-24,25-pentols were synthesized by reduction with LiAlH(4) of the corresponding epoxides prepared from (24S)- or (24R)-27-nor-5beta-cholest-25-ene-3alpha, 7alpha,12alpha,24-tetrol. The stereochemistries at C-25 were deduced by comparison of the C(26)-24,25-pentols with the oxidation products of (24Z)-27-nor-5beta-cholest-24-ene-3alpha,7alpha, 12alpha-triol with osmium tetraoxide. On the basis of this assignment, the principal bile alcohol excreted into human and rat urine was determined to be (24S,25R)-27-nor-5beta-cholestane-3alpha,7alpha, 12alpha,24,25-pentol, accompanied by a lesser amount of (24R, 25R)-isomer. To elucidate the biosynthesis of the C(26)-24,25-pentol, a putative intermediate, 3alpha,7alpha, 12alpha-trihydroxy-27-nor-5beta-cholestan-24-one, derived from 3alpha,7alpha, 12alpha-trihydroxy-24-oxo-5beta-cholestanoic acid by decarboxylation during the side-chain oxidation of 3alpha,7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid, was incubated with rat liver homogenates. The 24-oxo-bile alcohol could be efficiently reduced to yield mainly (24R)-27-nor-5beta-cholestane-3alpha,7alpha, 12alpha,24-tetrol. If a 25R-hydroxylation of the latter steroid occurs, it should lead to formation of (24S,25R)-C(26)-24,25-pentol. Now it has appeared that a major bile alcohol excreted into human urine is (24S,25R)-27-nor-5beta-cholestane-3alpha,7alpha, 12alpha, 24, 25-pentol, which might be derived from 3alpha,7alpha, 12alpha-trihydroxy-27-nor-5beta-cholestan-24-one via (24R)-27-nor-5beta-cholestane-3alpha, 7alpha,12alpha,24-tetrol.     

Vitamin A and glutathione-mediated free radical damage: competing reactions with polyunsaturated fatty acids and vitamin C

Vitamin A (retinol reacts extremely rapidly (k = 1.4 x 10(9) M-1 s-1) with thiyl free radicals derived from glutathione to form a free radical with a very strong visible absorption (lambda max. = 380 nm, E max. = 4.0 x 10(4) M-1 cm-1). Arachidonate, linolenate, linoleate and ascorbate also react readily but much more slowly (k = 2.2 x 10(7), 1.9 x 10(7), 1.3 x 10(7) and 3.6 x 10(8) M-1 s-1 respectively). These results support the possibility that vitamin A might play a role in protecting lipid membranes against thiyl free radical mediated damage.     

Rat liver contains a novel nucleotide, Ypp5'A2'p, related to adenosine 2',5'-bisphosphate

A novel nucleotide, Ypp5'A2'p, has been purified through perchloric acid extraction of rat liver followed by DEAE-cellulose and ion pair high pressure liquid chromatographies. Y stands for an unknown compound, probably a nucleoside, whose sugar moiety is different to beta-D (deoxy) ribose. Treatment of Ypp5'A2'p with snake venom phosphodiesterase renders Yp and adenosine 2',5'-bisphosphate (pAp). After elimination of the terminal phosphate with alkaline phosphatase, the resulting nucleotide (Ypp5'A) yielded Yp and 5'-AMP when hydrolyzed by the phosphodiesterase. The following ultraviolet absorption spectral characteristics were determined at pH 7: Ypp5'A2'p (lambda max = 265 nm; A250/A260 = 0.76; A280/A260 = 0.79); Yp (lambda max = 279 nm; A250/A260 = 0.70; A280/A260 = 1.70). The molar extinction coefficient found for Yp at 280 nm was 20.6 x 10(3) M-1 cm-1.     

Production of 10-oxo-19-nor-25-hydroxyvitamin D3 by solubilized kidney mitochondria from chick and rat

Cholate-solubilized chick kidney mitochondria that 1-hydroxylated 25-hydroxyvitamin-D3 (25-OH-D3) upon reconstitution also produced 10-oxo-19-nor-25-OH-D3, which co-eluted with 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) on normal phase high performance liquid chromatography (HPLC) with hexane:propanol-2 (9:1), the traditional chromatographic system for isolating 1,25-(OH)2-D3. The 10-oxo derivative was separated from 1,25-(OH)2-D3 by normal phase HPLC with dichloromethane:propanol-2 (19:1) or by reverse phase HPLC with methanol:water (4:1). Unlike 1,25-(OH)2-D3 production, formation of 10-oxo-19-nor-25-OH-D3 did not require a source of reducing equivalents and was blocked by the antioxidants, diphenyl-rho-phenylenediamine, and butylated hydroxytoluene, implicating a free radical or peroxidative synthetic mechanism. Rat kidney mitochondria solubilized with cholate or with cholate and Emulgen 911 produced 10-oxo-19-nor-25-OH-D3 but no detectable 1 alpha,25-(OH)2-D3. These results stress the importance of careful identification of vitamin D metabolites produced in vitro and suggest the use of alternate chromatographic conditions for isolating 1,25-(OH)2-D3 or inclusion of antioxidants in the assay of solubilized 1 alpha-hydroxylase to eliminate contamination of 1,25-dihydroxyvitamin D3 with 10-oxo-19-nor-25-OH-D3.     

A pulse-radiolysis study of the reaction of hydrated electrons with N'-formylkynurenine and related compounds: electron-transfer reactions with nucleic acid components.

The reactivity of N'-formylkynurenine (FK) derivatives towards eaq has been investigated. The reduced transient species have been characterized (lambda max approximately 340, 440 nm, epsilon lambda max approximately 3000-1000 M-1 cm-1, pKa approximately 7.8). Owing to the strong FK electron affinity, electron-transfer reactions occur from purine (except guanine) and pyrimidine electron adducts to FK (k approximately 2-7 x 10(9) M-1 s-1). As some FK derivatives bind to DNA (or polynucleotides) the protective effect of complexation on FK-DNA (or polynucleotides) adduct formation has been investigated.     

Inhibition of biofilm formation and swarming of Escherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone

The quorum-sensing disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) of the alga Delisea pulchra was found to inhibit the swarming motility of Escherichia coli completely at 13 microg cm-2 (also at 20 microg ml-1) but did not inhibit its growth rate at 13-52 microg cm-2 or from 20 to 100 microg ml-1. Swimming was not inhibited by the furanone at 20-40 microg ml-1. In addition, confocal scanning laser microscopy revealed that this furanone at 60 microg ml-1 inhibited the biofilm formation of E. coli, as it decreased its thickness by 55%, reduced the number of water channels and decreased the percentage of live cells by 87%. This suggests that natural furanone may be used as a new method to control bacterial biofilms that does not involve toxicity. Furanone at 10 microg ml-1 also inhibited by 3300-fold the quorum sensing of Vibrio harveyi via autoinducer 1 (AI-1) and inhibited by 5500-fold that of V. harveyi via of autoinducer 2 (AI-2) as well as inhibited by 26-600-fold the quorum sensing of E. coli via AI-2; hence, this furanone is a non-specific intercellular signal antagonist.     

Characterization of indo-1 and quin-2 as spectroscopic probes for Zn2(+)-protein interactions

1-[2-Amino-5-(6-carboxyindol-2-yl)phenoxyl]-2-(2'- amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (indo-1) and 2-[2-(bis(carboxymethyl)amino-5-methylphenoxy) methyl]-6- methyl-8-[bis-(carboxymethyl)amino]quinoline (quin-2) are sensitive, spectral indicators for Zn2+. Additions of subsaturating Zn2+ to 10-80 microM indo-1 or quin-2 at pH 7.0 produce uv difference spectra with isosbestic wavelengths at 342 and 282 nm or at 342, 317, and 252 nm, respectively. Formation of 1:1 Zn2+:indicator complexes at pH 7.0 and 20 degrees C in the absence (presence) of 100 mM KCl gives delta epsilon max = -2.4 +/- 0.2 X 10(4) M-1 cm-1 at 367 nm (-2.1 +/- 0.2 X 10(4) M-1 cm-1 at 365 nm) for indo-1 and delta epsilon max = -2.7 +/- 0.1 X 10(4) M-1 cm-1 at 266 nm (-2.6 +/- 0.1 X 10(4) M-1 cm-1 at 265 nm) for quin-2. Competition experiments at pH 7.0 and 20 degrees C with indo-1 and quin-2 and also 4-(2-pyridylazo)resorcinol (PAR) as the second chelator in the absence (presence) of 100 mM KCl yield apparent affinity constants: K'A = 2.5 +/- 1.0 X 10(10) M-1 (6.2 +/- 0.5 X 10(9) M-1) for indo-1 binding Zn2+ and K'A = 9.4 +/- 3.3 X 10(11) M-1 (2.7 +/- 0.1 X 10(11) M-1) for quin-2 binding Zn2+. The above constants provide the basis for rapid steady-state spectrophotometric determinations of the affinity of a protein for Zn2+ with K'A approximately 10(10) - 10(13) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)