Interaction of monosulfonate tetraphenyl porphyrin, a competitive inhibitor, with acetylcholinesterase

Monosulfonate tetraphenyl porphyrin (TPPS(1)) forms a 1:1 complex with electric eel acetylcholinesterase (AChE) inducing a loss in TPPS(1) absorbance at 402 nm and the appearance of a new absorbance centered at 442 nm. In the presence of AChE, the fluorescence of TPPS(1) at 652 nm is slightly narrowed, with the maximal 652 nm fluorescence shifted from 407 to 412 nm excitation wavelength. The fluorescence peak of TPPS(1) at 712 nm shifts to 716 nm in the presence of AChE. TPPS(1) is a competitive inhibitor of AChE. The addition of acetylcholine iodide (AChI) or the competitive inhibitor tetracaine to the preformed AChE-TPPS(1) complex results in a loss of the 442 nm absorbance band as the porphyrin is displaced from AChE. The absorbance peak does not decrease in the presence of procaine, a non-competitive inhibitor.     

Extended lifetime of reagentless detector for multiple inhibitors of acetylcholinesterase

Competitive inhibitors of acetylcholinesterase (AChE) are detected using an evanescent wave technique to monitor changes in the absorbance spectrum of an AChE-monosulfonate tetraphenyl porphyrin (TPPS(1)) complex immobilized on the surface of a glass slide. In this technique, porphyrin is displaced from the AChE active site by the inhibitor. The loss in absorbance intensity of the characteristic absorbance peak for the AChE-TPPS(1) complex at 446 nm is linearly dependent on the log of the inhibitor concentration. This technique yields detection limits at 3:1 S/N of 37 ppt for eserine, 50 ppt for galanthamine, 100 ppt for scopolamine, 250 ppt for tetracaine, 45 ppt for diazinon, and 83 ppb for Triton X-100. When stored under vacuum, the enzymatic lifetime of the immobilized AChE surface is greater than 73 days while the responsive lifetime of the immobilized AChE-TPPS(1) surface is currently 49 days.     

Spectrophotometric detection of pentachlorophenol (PCP) in water using immobilized and water-soluble porphyrins

The spectrophotometric properties of porphyrins are altered upon interaction with chlorophenols and other organochlorine pollutants. Meso-tetra(4-sulfonatophenyl)porphyrin (TPPS), zinc meso-tetra(4-sulfonato phenyl)porphyrin (Zn-TPPS), monosulfonate-tetraphenylporphyrin (TPPS1), meso-tri(4-sulfonatophenyl)mono(4-carboxyphenyl)porphyrin (C1TPP), meso-tetra(4-carboxyphenyl)porphyrin (C4TPP), and copper meso-tetra(4-carboxyphenyl)porphyrin (Cu-C4TPP) in solution exhibit a broad absorbance in the range 400-450 nm Soret region. The interaction of the above mentioned porphyrins in solution with pentachlorophenol (PCP) induces a red shift in the Soret spectrum with absorbance losses at 413, 418, 403, 405, 407, and 404 nm, respectively, and the appearance of new peaks at 421, 427, 431, 416, 417, and 416 nm, respectively. The intensity of the Soret spectral change is proportional to the pentachlorophenol concentration with a detection limit of 1, 0.5, 1.16, 1, 0.5, and 0.5 ppb, respectively. The interaction of (C4TPP) and (Cu-C4TPP) in solution with PCP shows to concentration dependent for concentrations less than 4 ppb the dependence was log-linear. However, for concentrations greater than 4 ppb the relation was linear. Monosulfonate-tetraphenylporphyrin immobilized as a monolayer on a Kimwipe tissue exhibits an absorbance peak in the Soret region at 422 nm. The interaction of the porphyrin with PCP induces a red shift in the Soret spectrum with absorbance loss at 419 nm and the appearance of new peaks at 446 nm. The intensity of the Soret spectral change is proportional to the log of PCP concentration. The detection limit with immobilized TPPS1 for PCP is 0.5 ppb. These results suggest the potential for development of spectrophotometric chemosensor for PCP residues in water with detection limits less than US EPA maximum contaminate level (MCL) of 1 ppb. The immobilized TPPS1 on the Kimwipe will make it possible to develop a wiping sensors to monitor the PCP or other pesticides residues on the vegetables or wood products.     

Reagent-less detection of a competitive inhibitor of immobilized acetylcholinesterase

The interaction of monosulfonate tetraphenyl porphine (TPPS(1)) with immobilized acetylcholinesterase (AChE) yields a characteristic absorbance peak at 446 nm. Addition of acetylcholine iodide or the competitive inhibitor tetracaine to the immobilized TPPS(1)-AChE complex results in a decrease in absorbance intensity at 446 nm due to displacement of the porphyrin from the active site. The loss in intensity at 446 nm is linearly dependent on tetracaine concentration at levels below 100 ppb. Tetracaine concentrations as low as 300 ppt have been detected.     

An Anionic Porphyrin Binds <Emphasis Type="Italic">β</Emphasis>-Lactoglobulin A at a Superficial Site Rich in Lysine Residues

Binding of small ligands to globular proteins remains a major research topic in biophysics. We have studied the binding of several photoactive dyes to β-lactoglobulin (BLG), as a model to investigate the photoinduced effects of porphyrins on proteins. A combination of optical spectroscopies (fluorescence, circular dichroism) and molecular docking simulations were used to estimate the pH-dependence of the binding parameters and the docking location for meso-tetrakis(sulfonatophenyl)-porphyrin (TPPS). We have observed that the binding of TPPS is not modulated by the pH-mediated conformational transition of the protein (i.e., Tanford transition). Binding of TPPS appears to occur with some degree of negative cooperativity. Moreover, TPPS remains bound even upon partial denaturation of the protein. These results are consistent with a superficial binding site at a location removed from the aperture of the interior β-barrel. Binding occurs through electrostatic interactions between the negative SO₃ ⁻ groups of TPPS and positively charged Lys and Arg residues. This is the first study that explores the interaction of an anionic porphyrin with BLGA in a pH range that spans across the Tanford transition. Establishing the location of the binding site will enable us to explain the photoinduced conformational effects mediated by TPPS on BLG. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Noncovalent interactions of peptides with porphyrins in aqueous solution: conformational study using vibrational CD spectroscopy.

Noncovalent interactions of poly(L-lysine) (PL), oligopeptides L-lysyl-L-alanyl-L-alanine and (L-lysyl-L-alanyl-L-alanine)(2) with meso-tetrakis(4-sulfonatophenyl)porphine (TPPS), and poly(L-glutamic acid) (PLGA) with meso-tetrakis(1-methyl-4-pyridyl)porphine tetra-p-tosylate (TMPyP) in aqueous solutions have been studied using combination of spectroscopic methods: Vibrational circular dichroism (VCD) spectroscopy in the mid-infrared region provides a direct information on conformational changes of the polypeptides and oligopeptides caused by interactions with porphyrins; ultraviolet-visible absorption, fluorescence, and electronic circular dichroism (ECD) reveal the aggregation characterization of the porphyrin part of the complexes. Interactions of TPPS with tripeptide, hexapeptide, and PL containing about ten amino acid residues in the molecular chain are accompanied with the changes of VCD patterns in the amide I' region. In these cases, the conformation of the oligopeptide part of complexes is obviously influenced by interactions with TPPS and partial changes of random coil structure are observed in VCD. When PL was composed of the hundreds of lysine residues, just a weak intensity decrease was detected and the shape of VCD spectrum typical for the random coil structure was preserved. As follows from the uv-vis absorption and fluorescence spectra, porphyrin molecules are attached to peptides by electrostatic interaction as a monomer or dimer and interaction between porphyrin and peptide depends on the polypeptide chain length. For the PLGA-TMPyP system with PLGA containing from tens to hundreds of glutamic acid residues in the chain, the VCD spectra were unchanged when TMPyP was presented in the aqueous solution of PLGA and random coil conformation of PLGA-TMPyP aggregates was preserved.     

Interaction of 3-Alkylpyridinium Polymers from the Sea Sponge Reniera sarai with Insect Acetylcholinesterase

3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS–PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS–PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.     

Interaction of 3-Alkylpyridinium Polymers from the Sea Sponge Reniera sarai with Insect Acetylcholinesterase

3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS–PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS–PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.     

Conformational transitions in protein-protein association: binding of fasciculin-2 to acetylcholinesterase

The neurotoxin fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex.     

Deuterium exchange on micrograms of proteins by attenuated total reflection Fourier transform infrared spectroscopy on silver halide fiber.

We illustrate the use of polycrystalline silver halide fibers (2-20 microns transparency range) for attenuated total internal reflection Fourier transform infrared (IR) spectroscopic measurements of microsamples (10 micrograms of protein). A powerful adjunct technique is a simple method for carrying out deuterium for proton exchange. Spectra of trypsin, soybean trypsin inhibitor, and their complex are easily obtained. Two kinds of difference spectra (DS) are revealing: DS1 (changes in protein on combination with ligand), IR of the trypsin-soybean trypsin inhibitor complex (T.SBTI complex)--sigma [IR of trypsin (T) + IR of soybean trypsin inhibitor (SBTI)], the small values at all wavelengths indicating no conformational change of the proteins upon complexation, and DS2 (changes in materials on deuteration), IR of protioprotein--IR of deuterioprotein, which reveals the infrared bands affected by deuteration. The rate and the extent of the exchange are additional valuable parameters readily measured with this technique. In the present instance, the rate and the amount of the exchange for T.SBTI complex after 30 min was substantially less than that expected from the simple sum of the same parameters for the two individual proteins, T and SBTI. The enzymatic activity of trypsin on the fiber survived for more than a day, no autodegradation being detected by SDS-gel electrophoresis.     

Crystal structures of the PNA-porphyrin complex in the presence and absence of lactose: mapping the conformational changes on lactose binding, interacting surfaces, and supramolecular aggregations

The extraordinary recognition specificity of lectins for carbohydrate ligands appears to be violated as they also bind to porphyrins and other noncarbohydrate ligands. In this study, crystal structures of meso-tetrasulfonatophenylporphyrin (H(2)TPPS) bound to peanut agglutinin (PNA) in the presence and absence of lactose were determined. The binding of H(2)TPPS with PNA involved 11 molecules of H(2)TPPS in different supramolecular stacking arrangements associated with a tetramer of PNA in the crystals of the PNA-H(2)TPPS binary complex as well as the PNA-H(2)TPPS-lactose ternary complex. The ternary complex involved lactose binding only to two subunits of the PNA tetramer, which did not have porphyrin interacting in the vicinity of the carbohydrate-binding site. Comparison of the two structures highlighted the plasticity of the carbohydrate-binding site expressed in terms of the conformational change in lactose binding. The unusual quaternary structure of PNA, which results in exposed protein-protein interaction sites, might be responsible for the porphyrin binding. The association of porphyrin in diverse oligomeric stacking arrangements observed in the PNA-H(2)TPPS complex suggested the possibility of protein-porphyrin aggregation under abnormal physiological conditions. The structures described here provide a possible native conformation of the carbohydrate-binding site of PNA in the absence of the ligand, highlight mapping of the unsaturated binding surfaces of PNA using porphyrin interactions, indicate new leads toward possible application of this lectin in photodynamic therapy, and exhibit diverse modes of porphyrin-lectin interactions with implications to porphyria, a disease that results from abnormal accumulation of porphyrins.     

Porphyrins affect the self-assembly of tubulin in solution

Self-assembly of tubulin heterodimers in solution has been studied in the past to predict the effects that ligands and/or conformational changes have on the formation of tubulin filaments. Self-assembly of tubulin in solution has produced formations similar to cellular microtubules (MTs). The present study reports on the effects that two porphyrins (protoporphyrin IX, PPIX and tetrakis(4-sulfonatophenyl)porphyrin, TPPS) produce on the self-assembly of tubulin α,β-heterodimers in buffer solution. The study shows that, when incubated simultaneously with MT-stabilizing ligands (i.e., paclitaxel and guanosine triphosphate, GTP), porphyrins do not affect the ability of tubulin to form MT. However, if paclitaxel and GTP are added after tubulin has been allowed to self-assemble in the presence of either porphyrin, the ability to form MT-like structures is reduced or suppressed. We suggest that this effect is due to the formation of porphyrin-mediated aggregates that cannot be broken or elongated by the addition of GTP or paclitaxel.     

A simple spectrophotometric method for the measurement of ribonuclease activity in biological fluids

We have developed a rapid and sensitive method for detecting ribonuclease (RNAase). The method makes use of a RNa-Pyronine Y complex which has a different absorption spectrium from that of Pyronine Y alone. When the RNA is hydrolyzed by RNAase, the spectrum of the complex changes to that of unbound Pyronine Y. The resultant decrease in absorbance at 572 nm is linear for final RNAase concentrations ranging from 2 to 45 ng/ml. Optimal assay conditions were 11.5 μg/ml Pyronine Y, 0.56 mg/ml RNA, 80 μmol/ml Tris-HCl buffer, pH 7.8 and 2–45 ng/ml RNAase. The effect of complex concentration, PH, molarity and temperature upon the rate of the reaction were determined.The assay is applicable to crude cell-free extracts.     

An allosteric model for ribonuclease.

Data from two assay systems show that the kinetics of the hydrolysis of cytidine 2':3'-cyclic monophosphate by bovine pancreatic RNAase (ribonuclease) is not consistent with conventional models. An allosteric model involving a substrate-dependent change in the equilibrium between two enzyme conformations is proposed. Such a model gives rise to a calculated curve of velocity versus substrate concentration which fits the experimental data. The model is also consistent with the results of an examination of the tryptic digestion of RNAase. Substrate analogues are able to protect RNAase against hydrolysis by trypsin and the percentage of RNAase activity which remains after digestion increases sigmoidally as the analogue concentration is increased. The model also explains the pattern seen in the Km values quoted in the literature and is consistent with strong physical evidence for a ligand-induced conformational change for RNAase reported in the literature.     

Heat stabilization produced by protein-protein association. A differential scanning calorimetric study of the heat denaturation of the trypsin-soybean trypsin inhibitor and trypsin-ovomucoid complexes.

The irreversible thermal denaturation of the association complexes of bovine beta-trypsin with soybean trypsin inhibitor or ovomucoid was observed with a differential scanning calorimeter. Association of trypsin with either inhibitor results in increased heat stability. The largest effect is observed with beta-trypsin and soybean trypsin inhibitor. At pH 6.7, first order rate constants (s-1) for denaturation at 72 degrees, determined at a heating rate of 10 degrees per min, are: beta-trypsin, 30 times 10-3; soybean trypsin inhibitor, 9 times 10-3; trypsin-soybean trypsin inhibitor complex, 0.4 times 10-3. Under equivalent conditions, rate constants for ovomucoid and trypsin-ovomucoid complex are 4 times 10-3 and 1 times 10-3 s-1, respectively. These changes in rate correspond to heat stabilization of trypsin equivalent to an increase of 16 and 9 degrees, respectively, in its observed denaturation temperature. Rate constants determined for beta-trypsin and trypsin-soybean trypsin inhibitor complex are independent of heating rate; those for soybean trypsin inhibitor and ovomucoid are a function of heating rate. This suggests that predenaturational conformational alterations may be important steps in the denaturation of the inhibitors. Activation energies for denaturation of the complexes and their components are all similar, averaging 70 kcal per mol. The large activation energies observed suggest that denaturation of the complexes is not rate-limited by their dissociation.     

Assignment of the natural abundance 13C spectrum of proteins using 13C 1H-detected heteronuclear multiple-bond correlation NMR spectroscopy: structural information and stereospecific assignments from two- and three-bond carbon-hydrogen coupling constants.

Proton-detected heteronuclear multiple-bond 1H-13C correlations (HMBC) previously have been used for assignment purposes in a variety of isotopically enriched proteins. In the present study it is demonstrated that the technique yields an almost complete assignment of the natural abundance 13C spectrum of the protein basic pancreatic trypsin inhibitor (BPTI). In addition, the technique permits additional 1H assignments to be made for this well-studied protein. The intensities of observed correlations permit rough estimates to be made of 2J(C,H) and 3J(C,H) coupling constants. These couplings can be used for conformational studies of both the side chains and the backbone. Intra- and interresidue coupling between C alpha H and the carbonyl carbon provides information about the backbone angles psi and phi. Side-chain conformations can be determined from both two- and three-bond carbon-hydrogen coupling constants. The present study of BPTI together with its known high-precision solution structure yields an experimental correlation between resonance intensities and secondary structure. The spectra show the potential of the method in analyzing 13C NMR spectra of nonenriched proteins. The method yields 13C NMR chemical shifts, which are versatile parameters to be used to monitor structural changes, titrations, etc.     

Raman studies of the conformation of the basic pancreatic trypsin inhibitor.

The vibrational Raman spectra of the basic pancreatic trypsin inhibitor in aqueous solution, as lyophilized powder and in a single crystal and presented. The thermal stability of this protein is demonstrated by the fact that minor alterations in the spectrum, mainly in the amide III band near 1260 cm-1, occur in the solution spectrum only at temperatures above 75 degrees C. No significant spectral changes appear when the pH value of the solution is varied in the range from 1.5 to 8.7. The distinct differences of the powder spectrum compared to that of the solution, show that lyophilization causes appreciable conformational changes both in the main-chain and in the side-chains. A difference in main chain conformation of the basic pancreatic trypsin inhibitor in single crystal and in solution is suggested by different amide III frequencies.     

The time dependent UV resonance Raman spectra, conformation, and biological activity of acetylcholine analogues upon binding to acetylcholine binding proteins.

In order to obtain quantitative data on the relation between the conformation of acetylcholine and its interaction with biologically significant proteins, a series of acetylcholine analogues with absorption bands in the region 200-300 nm have been synthesized or obtained commercially. Each of these compounds were assayed to measure its activity as an ion channel activator of the nicotinic acetylcholine receptor protein (AChR). In addition, the suitability of some of these compounds as substrates for hydrolysis by acetylcholine esterase (AChE) was determined. One of these analogues, dimethylthionocarbamylcholine (DMTC-Ch), has the ester carbonyl oxygen replaced by a thionyl sulfur. DMTC-Ch has been found to be quite active as an ion channel activator when bound to AChR and was found to react with the enzyme AChE as a suicide substrate. It forms a thionoester of the serine at the AChE active site by an ester exchange reaction that releases the choline as the first product. However, the second or acid product is not released even at pH 7.5 over a period of days. This acetylcholine analog has an absorption band at about 240 nm and exhibits very strong ultraviolet resonance Raman (UVRR) spectra using 239 nm excitation from a frequency modified Nd:YAG laser. This technique allows observation of both conformational changes of the ligand molecule that result in frequency changes as well as changes in the excited state electronic structure that results in changes in the relative intensity of the Raman bands. The time dependence of the UVRR spectrum of the ligand upon binding to both AChE and AChR has been studied from 0.1 msec to minutes. Some time dependence in the conformation of DMTC-Ch upon binding to AChE has been found for very short (0.1-0.5 msec) times. However, no change in the conformation of this neurotransmitter analog is found in the available time range upon binding to AChR. From these data it is concluded that a previous suggestion that acetylcholine has a conformational change upon binding to AChR may be incorrect since the solution behavior of the carbamyl cholines and acetylcholine are similar. Even if acetylcholine does change conformation upont binding to AChR, it is unlikely that such a conformational change plays a significant role in channel activation. We present strong evidence that acetylcholine and its analogues can be active in a variety of conformations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biological effects of anionic meso-tetrakis (para-sulfonatophenyl) porphyrins modulated by the metal center. Studies in rat liver mitochondria

In this paper, we present a study about the influence of the porphyrin metal center and meso ligands on the biological effects of meso-tetrakis porphyrins. Different from the cationic meso-tetrakis 4-N-methyl pyridinium (Mn(III)TMPyP), the anionic Mn(III) meso-tetrakis (para-sulfonatophenyl) porphyrin (Mn(III)TPPS4) exhibited no protector effect against Fe(citrate)-induced lipid oxidation. Mn(III)TPPS4 did not protect mitochondria against endogenous hydrogen peroxide and only delayed the swelling caused by tert-BuOOH and Ca²⁺. Fe(III)TPPS4 exacerbated the effect of the tert-BuOOH, and both porphyrins did not significantly affect Fe(II)citrate-induced swelling. Consistently, Fe(III)TPPS4 predominantly promotes the homolytic cleavage of peroxides and exhibits catalytic efficiency ten-fold higher than Mn(III)TPPS4. For Mn(III)TPPS4, the microenvironment of rat liver mitochondria favors the heterolytic cleavage of peroxides and increases the catalytic efficiency of the manganese porphyrin due to the availability of axial ligands for the metal center and reducing agents such as glutathione (GSH) and proteins necessary for Compound II (oxomanganese IV) recycling to the initial Mn(III) form. The use of thiol reducing agents for the recycling of Mn(III)TPPS4 leads to GSH depletion and protein oxidation and consequent damages in the organelle.     

Effect of chloroquine wash-out period of photosensitizers in the skin and selected organs in rats

In the last decade, photodynamic therapy has become an alternative method for the diagnosis and therapy of tumors. In human medicine hematoporphyrin derivatives, sulfonated hydrophilic meso-tetraphenylporphyrin (TPPS4) and an oligomer of hematoporphyrin (Photosan 3), are widely used. Chloroquine is used for the treatment of porphyria cutanea tarda for its power to release porphyrins from the liver tissue. The kinetics of two porphyrin photosensitizers TPPS4 and Photosan 3 in the skin and some organs as well as the effect of chloroquine on the porphyrin excretion and their accumulation in skin and organs of Wistar rats were studied. TPPS4 exhibited maximum fluorescence in skin 48 h after application with decreasing to basal level from the 8th to the 14th day. Maximum fluorescence was reached at 72 hours after Photosan 3 application and it decreased to basal level during 96 hours after application. TPPS4 caused significantly higher fluorescence compared to Photosan 3. Chloroquine after oral administration did not change the fluorescence of skin, but it significantly decreased the TPPS4 concentration in rat organs if chloroquine treatment started 3 days or 2 weeks after TPPS4 application. Chloroquine significantly decreased the serum TPPS4 concentration during the period of 28 days. Fluorescence of skin was significantly higher and lasted longer after application of TPPS4 compared to Photosan 3. Chloroquine after oral administration did not influence the fluorescence of the skin, but it significantly decreased the TPPS4 concentration in rat organs. This effect could be useful in photodynamic therapy for mobilizing exogenous porphyrins from tissues after parenteral photodynamic therapy.