Characterization of the interaction between prostacyclin and human serum albumin using a fluorescent analogue, 2,6-dichloro-4-aminophenol iloprost

We synthesized a fluorescent probe, 2,6-dichloro-4-aminophenol iloprost or dichlorohydroxyphenylamide of iloprost (DCHPA-iloprost) by reacting the stable prostacyclin analog, iloprost (ZK 35 374), with 2,6-dichloro-4-aminophenol with a yield of 60%. This probe exhibited an optical spectrum which overlapped with the emission spectrum of the sole tryptophan of human serum albumin (HSA). Energy transfer from the tryptophan residue to the phenol moiety of DCHPA-iloprost was observed. We utilized this donor-quenching phenomenon to quantitate the binding stoichiometry and affinity as well as the association rate of DCHPA-iloprost binding to HSA. As DCHPA-iloprost showed similar binding characteristics similar to those of iloprost and prostacyclin and competed with iloprost for HSA binding sites, we used DCHPA-iloprost as a probe to locate the binding domain of prostacyclin (PGI2) in HSA. The distance between the tryptophan indole and the phenol group of DCHPA-iloprost was estimated to be 15-18 A. Because iloprost binding to HSA was competitive with warfarin and not with free fatty acid, we propose that PGI2 binds to the 'domain 2' of HSA was competitive with warfarin and not with free fatty acid, we propose that PGI2 binds to the 'domain 2' of HSA molecules. A possible molecular mechanism by which HSA reduces the chemical degradation of PGI2 and stabilizes its activity could be derived from this model.     

Analysis of tryptophan fluorescence lifetimes in a series of human serum albumin mutants with substitutions in subdomain 2 A

Tryptophan 214, the only tryptophan residue in human serum albumin, is located in the physiologically important subdomain 2A ligand binding site. In the present study the fluorescence lifetime of tryptophan 214 in the following human serum albumin (HSA) mutants with substitutions in subdomain 2A were determined: K195M, K199M, F211V, R218M, R218H, R218A, R222M, H242V, and R257M. An HSA mutant in which tryptophan was moved from subdomain 2A to subdomain 3A (W214L/Y411W) was also examined. Additionally, the fluorescence lifetime of tryptophan 214 in an HSA fragment consisting of subdomains 1A, 1B, and 2A (1A-1B-2A HSA) was determined. For those species expected to have the most dramatic changes in tryptophan microenvironment, W214L/Y411W and 1A-1B-2A HSA, clear changes in tryptophan lifetimes were observed. Significant changes were also seen for those species with mutations at position 218, which is next to tryptophan in the X-ray structure of HSA. However, significant changes were also observed for H242V and R257M, which contain substitutions at positions not immediately adjacent to tryptophan 214, highlighting the conformational flexibility of subdomain 2A.     

Spatial relationship between the prodan site,Trp-214, and Cys-34 residues in human serum albumin and loss of structure through incremental unfolding

Prodan (6-propionyl-2-(dimethylamino)-naphthalene), a competitive inhibitor of warfarin binding to human serum albumin (HSA) at drug site I, was used to determine the inter- and intradomain distances of HSA. The fluorescence resonance energy transfer (FRET) distances between prodan and Trp-214, prodan and 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CM)-modified Cys-34, and Trp-214 and CM-Cys-34 were determined to be 25.5 +/- 0.5 A, 33.1 +/- 0.8 A, and 32.4 +/- 1 A, respectively. FRET analysis showed that low concentration of palmitic acid (5 microM) increased the interdomain distance between the Trp-214 in domain II and CM-Cys-34 in domain I by approximately 5 A without perturbing the secondary structure of HSA and the immediate environment of Trp-214. Palmitic acid (5 microM) increased the prodan fluorescence by increasing the quantum yield of bound prodan without altering the tryptophan environment. However, palmitic acid (>10 microM) decreased the prodan fluorescence and increased the tryptophan fluorescence. Our results indicate that the high affinity palmitic acid binding site is located at the interface of domains I and II. On the basis of our measurements, a schematic model representing the drug site-1, Trp-214, and Cys-34 along with the palmitic acid sites has been constructed. In addition, prodan fluorescence, FRET, and ligand binding were used to monitor guanidine hydrochloride-induced denaturation of HSA. An analysis of the equilibrium unfolding data suggests that HSA undergoes a two-state unfolding transition with no detectable intermediate. However, kinetic analysis using multiple probes and thermal denaturation studies showed that the unfolding of the prodan site in HSA preceded the unfolding of tryptophan environment. In addition, the separation of domain I and II occurred before the global unfolding of the protein. The data support the idea that HSA loses its structure incrementally during its unfolding.     

The interaction of quercetin with human serum albumin: a fluorescence spectroscopic study

Quercetin (3,3',4',5,7-pentahydroxyflavone), a ubiquitous, bioactive plant flavonoid, is known to possess anti-cancer, anti-tumor, and other important therapeutic activities of significant potency and low systemic toxicity. In this communication, we report for the first time a study on the interactions of quercetin with the plasma protein human serum albumin (HSA), exploiting the intrinsic fluorescence emission properties of quercetin as a probe. Quercetin is weakly fluorescent in aqueous buffer medium, with an emission maximum at approximately 538 nm. Binding of quercetin with HSA leads to dramatic enhancement in the fluorescence emission intensity and anisotropy (r), along with significant changes in the fluorescence excitation and emission profiles. The excitation spectrum suggests occurrence of efficient F?rster type resonance energy transfer (FRET) from the single tryptophan-214 residue of HSA to the protein bound quercetin. The emission, excitation, and anisotropy (r=0.18 at [HSA]=30 microM) data (using the native protein) along with emission studies of quercetin using partially denatured HSA (by 8M urea) indicate that the quercetin molecules bind at a motionally restricted site near tryptophan-214 in the interdomain cleft region of HSA. Furthermore, the binding constant (K=1.9 x 10(5)M(-1)) and Gibbs free energy change (deltaG(0)=-30.12 kJ/mol)) for quercetin-HSA interaction have been calculated from the relevant anisotropy data. Implications of these results are examined, particularly in relation to prospective applications in biomedical research.     

Interaction of cromolyn sodium with human serum albumin: a fluorescence quenching study

The interaction between cromolyn sodium (CS) and human serum albumin (HSA) was investigated using tryptophan fluorescence quenching. In the discussion of the mechanism, it was proved that the fluorescence quenching of HSA by CS is a result of the formation of a CS–HSA complex. Quenching constants were determined using the Sterns–Volmer equation to provide a measure of the binding affinity between CS and HSA. The thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures were calculated. The distance r between donor (Trp²¹⁴) and acceptor (CS) was obtained according to fluorescence resonance energy transfer (FRET). Furthermore, synchronous fluorescence spectroscopy data and UV–vis absorbance spectra have suggested that the association between CS and HSA changed the molecular conformation of HSA and the electrostatic interactions play a major role in CS–HSA association.     

Binding of the bioactive compound 5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone to human serum albumin

5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone is one of the bioactive components isolated from Artemisia plants possessing antitumor therapeutic activities. In this paper, its binding properties and binding sites located on human serum albumin (HSA) have been studied using UV absorption spectroscopy, fluorescence spectroscopy and Fourier transform infrared (FT-IR) spectra. The results of fluorescence titration revealed that 5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone could strongly quench the intrinsic fluorescence of HSA by static quenching and there was only one class of binding sites on HSA for this drug. The binding constants at four different temperatures (289, 298, 310, and 318 K) were 1.93, 1.56, 1.22, and 0.93x10(5) L mol-1, respectively. The FT-IR spectra evidence showed that the protein secondary structure changed with reduction of alpha-helices about 27.6% at the drug to protein molar ratio of 3. The thermodynamic functions standard enthalpy change (DeltaH0) and standard entropy change (DeltaS0) for the reaction were calculated to be -18.70 kJ mol-1 and 36.62 J mol-1 K-1 according to the van't Hoff equation. These results and the molecular modeling study suggested that hydrophobic interaction was the predominant intermolecular force stabilizing the complex, and 5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone could bind to the site I of HSA (the Warfarin Binding site).     

Spectroscopic and Molecular Modeling Studies on the Interaction Between a Fluorine-Containing Triazole Derivative and Human Serum Albumin

The interaction between 4-(4-fluorobenzylideneamino)-5-propyl-4H-1,2,4-triazole-3-thiol (FBTZ) and human serum albumin (HSA) under simulative physiological conditions was investigated by fluorescence, UV–vis absorption and circular dichroism (CD) spectroscopy as well as molecular modeling method. Fluorescence spectroscopic data showed that the fluorescence quenching of HSA was a result of the formation of FBTZ–HSA complex. According to the modified Stern–Volmer equation, the effective quenching constants (K _a) of FBTZ to HSA were obtained at three different temperatures. The enthalpy change (ΔH) and entropy change (ΔS) were calculated on the basis of van′t Hoff equation, and the results showed that hydrogen-bonding and van der Waals forces were the dominant intermolecular forces to stabilize the complex. Site marker competitive replacement experiments demonstrated that the binding of FBTZ to HSA primarily took place in sub-domain IIA (Sudlow’s site I). The binding distance (r) between FBTZ and the tryptophan residue of HSA was estimated according to the theory of fluorescence resonance energy transfer. The conformational investigation showed that the presence of FBTZ induced some changes of secondary structure of HSA. Molecular modeling study further confirmed the binding mode obtained by experimental study.     

Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling

The binding of several different categories of small molecules to bovine (BSA) and human (HSA) serum albumins has been studied for many years through different spectroscopic techniques to elucidate details of the protein structure and binding mechanism. In this work we present the results of the study of the interactions of BSA and HSA with the anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS) monitored by fluorescence spectroscopy of the intrinsic tryptophans at pH 5.0. Similarly to pH 7.0 and 9.0, at low concentrations, the interaction of BSA with these surfactants shows a quenching of fluorescence with Stern-Volmer quenching constants of (1.1+/-0.1)x10(4) M(-1), (3.2+/-0.1)x10(3) M(-1) and (2.1+/-0.1)x10(3) M(-1) for SDS, HPS and CTAC, respectively, which are associated to the 'effective' association constants to the protein. On the interaction of these surfactants with HSA, an opposite effect was observed as compared to BSA, i.e., an enhancement of fluorescence takes place. For both proteins, at low surfactant concentrations, a positive cooperativity was observed and the Hill plot model was used to estimate the number of surfactant binding sites, as well as the association constants of the surfactants to the proteins. It is worthy of notice that the binding constants for the surfactants at pH 5.0 are lower as compared to pH 7.0 and 9.0. This is probably due to fact that the protein at this acid pH is quite compact reducing the accessibility of the surfactants to the hydrophobic cavities in the binding sites. The interaction of myristic acid with both proteins shows a similar fluorescence behaviour, suggesting that the mechanism of the interaction is the same. Recently published crystallographic studies of HSA-myristate complex were used to perform a modelling study with the aim to explain the fluorescence results. The crystallographic structure reveals that a total of five myristic acid molecules are asymmetrically bound in the macromolecule. Three of these sites correspond to higher affinity ones and correlate with high association constants described in the literature. Our models for BSA and HSA with bound SDS suggest that the surfactant could be bound at the same sites as those reported in the crystal structure for the fatty acid. The differences in tryptophan vicinity upon surfactant binding are explored in the models in order to explain the observed spectroscopic changes. For BSA the quenching is due to a direct contact of a surfactant molecule with the indole of W131 residue. It is clear that the binding site in BSA which is very close, in contact with tryptophan W131, corresponds to a lower affinity site, explaining the lower binding constants obtained from fluorescence studies. In the case of HSA the enhancement of fluorescence is due to the removal of static quenching of W214 residue in the intact protein caused by nearby residues in the vicinity of this tryptophan.     

Identification of Lys190 as the primary binding site for pyridoxal 5'-phosphate in human serum albumin.

The covalent binding of pyridoxal 5'-phosphate (PLP) to human serum albumin (HSA) is important in the regulation of PLP metabolism. In plasma, PLP is bound to HSA at a single high-affinity and at two or more nonspecific sites. To characterize the primary PLP binding site, HSA was incubated with [3H] PLP, and the Schiff base linkage was reduced with potassium borohydride. Tryptic peptides were purified, and the major labeled peptide was sequenced. Amino acid analysis confirmed a homogeneous peptide Leu-Asp-Glu-Leu-Arg-Asp-Glu-Gly-Xaa-Ala-Ser-Ser-Ala-Lys which corresponds to residues 182-195 of HSA. The data indicate that Lys190 is the primary PLP binding site. This Lys residue is distinct from other sites of covalent adduct formation; namely, the primary sites for nonenzymatic glycosylation (Lys525) and acetylation by aspirin (Lys199).     

Chromatographic analysis of carbamazepine binding to human serum albumin

In this study, high-performance affinity chromatography was used to characterize the binding of carbamazepine to an immobilized human serum albumin (HSA) column. Frontal analysis was first used to determine the association equilibrium constant and binding capacity for carbamazepine on this column at various temperatures. The non-specific binding of carbamazepine within the column was also considered. The results indicated that carbamazepine had a single binding site on HSA with an association equilibrium constant of 5.3 x 10(3)M(-1) at pH 7.4 and 37 degrees C. This was confirmed through zonal elution self-competition studies. The value of DeltaG for this reaction was -5.35 kcal/mol at 37 degrees C, with an associated change in enthalpy (DeltaH) of -6.45 kcal/mol and a change in entropy (DeltaS) of -3.56 cal/molK. The location of this binding region was examined by competitive zonal elution experiments using probe compounds with known sites on HSA. It was found that carbamazepine had direct competition with l-tryptophan, a probe for the indole-benzodiazepine site of HSA, but allosteric interactions with probes for the warfarin, tamoxifen and digitoxin sites. Changes in the pH, ionic strength, and organic modifier content of the mobile phase were used to identify the predominant forces in the carbamazepine-HSA interaction.     

Studies by biointeraction chromatography of binding by phenytoin metabolites to human serum albumin

Biointeraction studies based on high performance affinity chromatography were used to investigate the binding of human serum albumin (HSA) to two major phenytoin metabolites: 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH). This was initially examined by conducting self-competition zonal elution experiments in which m-HPPH or p-HPPH were placed in both the mobile phase and injected sample. It was found that each metabolite had a single major binding site on HSA. Competitive zonal elution experiments using l-tryptophan, warfarin, digitoxin, and cis-clomiphene as site-selective probes indicated that m-HPPH and p-HPPH were interacting with the indole-benzodiazepine site of HSA. The estimated association equilibrium constants for m-HPPH and p-HPPH at this site were 3.2 (+/-1.2)x10(3) and 5.7 (+/-0.7)x10(3)M(-1), respectively, at pH 7.4 and 37 degrees C. Use of these metabolites as competing agents for injections of phenytoin demonstrated that m-HPPH and p-HPPH had direct competition with this drug at the indole-benzodiazepine site. However, the use of phenytoin as a competing agent indicated that this drug had additional negative allosteric interactions on the binding of these metabolites to HSA. These results agreed with previous studies on the binding of phenytoin to HSA and its effects on the interactions of HSA with site-selective probes for the indole-benzodiazepine site.     

A fluorescence study of type I and type II receptors of bone morphogenetic proteins with bis-ANS (4, 4'-dianilino-1, 1'-bisnaphthyl-5, 5' disulfonic acid)

Crystallography studies on several members of the bone morphogenetic protein (BMP) receptors suggested that hydrophobic regions in these proteins play an important role in their structure and function. In the present study, the environment sensitive fluorescent probe 4, 4'-dianilino-1, 1'-bisnaphthyl-5, 5' disulfonic acid (bis-ANS) was used to study the hydrophobic regions of the extracellular domain of the type I and II receptors for bone morphogenetic proteins (ecBMPR-IB and ecBMPR-II). A single bis-ANS binding site per receptor molecule was found for both receptors, but the two receptors interacted with bis-ANS with distinctive characteristics. A significant shift in the emission maximum from 498 to 510 nm was detected when bis-ANS binds ecBMPR-IB, but a negligible change in the emission maximum was observed when the dye binds ecBMPR-II. Under identical reaction conditions, the maximum fluorescence intensities of the probe (I(max)) for the ecBMPR-IB and -II are 4.0 and 6.2 x 10(4) arbitrary units, respectively. The probe binds to ecBMPR-IB and -II with K(d)=11.0 and 17.5 microM, respectively. The bis-ANS modified site on both receptor types was not readily accessible to acrylamide quenching. Fluorescence energy transfer experiments further revealed close proximity between the tyrosine (in ecBMPR-IB) and the tryptophan residue (in ecBMPR-II) and the respective bis-ANS binding site in these receptors. The binding of bis-ANS did not alter the ligand binding activity of ecBMPR-IB, but enhanced that of ecBMPR-II. These results show that the bis-ANS-modified hydrophobic site on the ecBMPR-IB and -II molecules plays a different functional role.     

Cleavage of single- and double-stranded DNAs containing an abasic residue by Escherichia coli exonuclease III (AP endonuclease VI).

The Escherichia coli exonuclease III (AP endonuclease VI) is a DNA-repair enzyme that hydrolyzes the phosphodiester bond 5' to an abasic site in DNA. To study how the enzyme recognizes the abasic site, we used oligonucleotides containing a synthetic abasic site at any desired position in the sequence. We prepared oligonucleotides containing an abasic residue such as 2'-deoxyribosylformamide, 2'-deoxyribose, 1',2'-dideoxy ribofuranose or propanediol. Duplex oligonucleotides containing an abasic residue used in this study were cleaved on the 5' side of the abasic site by exonuclease III in spite of the varieties of the bases opposite and adjacent to the abasic site. In addition, we observed that the enzyme cleaved single-stranded oligonucleotides containing an abasic site on the 5' side of the abasic site. These findings suggest that the enzyme may principally recognize the DNA-pocket formed at an abasic site. The indole ring of the tryptophan 212 residue of the exonuclease III is probably intercalated to the abasic site. The tryptophan in the vicinity of the catalytic site is conserved in the type II AP endonuclease from various organisms.     

Binding of hypocrellin B to human serum albumin and photo-induced interactions

Molecular binding of hypocrellins to human serum albumin (HSA) needs to be further clarified considering the phototherapeutic potentials of hypocrellins to vascular diseases. In the current work, it was estimated that the binding constant of hypocrellin B (HB) to HSA was 2.28 x 10(4) M(-1). Furthermore, based on the fluorescence responses for both HB and the tryptophan of HSA, it was suggested that the binding of HB to HSA should be more specific rather than distributed randomly on the surface of HSA, which was also confirmed by photobleaching of the tryptophan via photosensitization of HB. Besides, it was found that both of the photo-bleaching of the tryptophan and the photo-oxidation of HB were principally oxygen-dependent, suggesting reactive oxygen species generated via the photosensitization of HB, instead of the free radicals of the photosensitizer (HB*-), play the most important role in photodynamic processes.     

Characterization of the interaction between nitrofurazone and human serum albumin by spectroscopic and molecular modeling methods

The interaction of nitrofurazone (NF) and human serum albumin (HSA) has been studied by fluorescence spectroscopy, FT-IR spectroscopy and molecular modeling methods. The results showed that the fluorescence of HSA was quenched by NF in a static quenching mechanism. Thermodynamic parameters revealed that hydrogen bonds and van der Waals force played the major role during the interaction. The calculated binding distance (r) indicated that the non-radioactive energy transfer came into being in the interaction between NF and HSA. HSA had a single class of binding site at Sudlow' site I in subdomain IIA for NF, which was verified by the displacement experiment. The molecular modeling study further confirmed the specific binding sites of NF on HSA, such as the interaction between N11 and N14 of NF with Lue 283 and Ser 287 predominately through hydrogen bonds. Three-dimensional fluorescence spectra indicated that the polarity around the tryptophan residues decreased and the conformation of HSA changed after adding NF. FT-IR spectra showed that NF could induce the polypeptides of HSA unfolding because it changed α-helix and β-sheet into β-turn and random structure of HSA.     

Mutational and pH studies of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase.

The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.     

Structural Changes of Human Serum Albumin Induced by Cadmium Acetate

The structural changes of human serum albumin (HSA) induced by the addition of cadmium acetate were systematically investigated using UV–vis absorption, circular dichroism (CD), synchronous, and three‐dimentional (3D) fluorescence methods. The fluorescence spectra suggested the formation of cadmium acetate–HSA complex. UV absorption result indicated that the interaction between cadmium acetate and HSA could lead to the alteration of the protein skeleton. The structural analysis according to CD method showed that the cadmium acetate binding altered HSA conformation with a major reduction of α‐helix, inducing a partial protein unfolding. Synchronous fluorescence spectra suggested that cadmium acetate was situated closer to tryptophan residue compared to tyrosine residues, making tryptophan residue locate in a more hydrophobic environment. 3D fluorescence demonstrated that cadmium acetate could induce the HSA aggregation and cause a slight unfolding of the polypeptide backbone of the protein.     

Fluorescence studies of ATP-diphosphohydrolase from Solanum tuberosum var. Desirée

Chemical modification of potato apyrase suggests that tryptophan residues are close to the nucleotide binding site. Kd values (+/- Ca2+) for the complexes of apyrase with the non-hydrolysable phosphonate adenine nucleotide analogues, adenosine 5'-(beta,gamma-methylene) triphosphate and adenosine 5'-(alpha,beta-methylene) diphosphate, were obtained from quenching of the intrinsic enzyme fluorescence. Other fluorescent nucleotide analogues (2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-diphosphate. 1,N6-ethenoadenosine triphosphate and 1,N6-ethenoadenosine diphosphate) were hydrolysed by apyrase in the presence of Ca2+, indicating binding to the active site. The dissociation constants for the binding of these analogues were calculated from both the decrease of the protein (tryptophan) fluorescence and enhancement of the nucleotide fluorescence. Using the sensitised acceptor (nucleotide analogue) fluorescence method, energy transfer was observed between enzyme tryptophans and ethene-derivatives. These results support the view that tryptophan residues are present in the nucleotide-binding region of the protein, appropriately oriented to allow the energy transfer process to occur.     

A probe for the mutagenic activity of the carcinogen 4-aminobiphenyl: synthesis and characterization of an M13mp10 genome containing the major carcinogen-DNA adduct at a unique site

The duplex genome of Escherichia coli virus M13mp10 was modified at a unique site to contain N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG8-ABP), the major carcinogen-DNA adduct of the human bladder carcinogen 4-aminobiphenyl. A tetradeoxynucleotide containing a single dG8-ABP residue was synthesized by reacting 5'-d(TpGpCpA)-3' with N-acetoxy-N-(trifluoracetyl)-4-aminobiphenyl, followed by high-performance liquid chromatography purification of the principal reaction product 5'-d(TpG8-ABPpCpA)-3' (yield 15-30%). Characterization by fast atom bombardment mass spectrometry confirmed the structure as an intact 4-aminobiphenyl-modified tetranucleotide, while 1H nuclear magnetic resonance spectroscopy established the site of substitution and the existence of ring stacking between the carcinogen residue and DNA bases. Both 5'-d(TpG8-ABPpCpA)-3' and 5'-d(TpGpCpA)-3' were 5'-phosphorylated by use of bacteriophage T4 polynucleotide kinase and were incorporated into a four-base gap uniquely positioned in the center of the recognition site for the restriction endonuclease PstI, in an otherwise duplex genome of M13mp10. In the case of the adducted tetranucleotide, dG8-ABP was located in the minus strand at genome position 6270. Experiments in which the tetranucleotides were 5' end labeled with [32P]phosphate revealed the following: the adducted oligomer, when incubated in a 1000-fold molar excess in the presence of T4 DNA ligase and ATP, was found to be incorporated into the gapped DNA molecules with an efficiency of approximately 30%, as compared to the unadducted d(pTpGpCpA), which was incorporated with 60% ligation efficiency; radioactivity from the 5' end of each tetranucleotide was physically mapped to a restriction fragment that contained the PstI site and represented 0.2% of the genome; the presence of the lesion within the PstI recognition site inhibited the ability of PstI to cleave the genome at this site; in genomes in which ligation occurred, T4 DNA ligase was capable of covalently joining both modified and unmodified tetranucleotides to the gapped structures on both the 5' and the 3' ends with at least 90% efficiency. Evidence also is presented showing that the dG8-ABP-modified tetranucleotide was stable to the conditions of the recombinant DNA techniques used to insert it into the viral genome.(ABSTRACT TRUNCATED AT 400 WORDS)