Type II Photooxidation Mechanism of Biomolecules using Chloro ( 5 , 10 , 15 , 20-Tetraphenylporphyrinato ) indium ( III ) as a Photosensitizer

Foi determinado o mecanismo de fotooxidação de albumina de soro bovino (BSA), L-triptofano (Trp) e células vermelhas do sangue (RBC) por cloro(5,10,15,20-tetrafenilporfirinato) de índio(III) (InTPP). A velocidade de fotooxidação de Trp, BSA e RBC por InTPP foi diminuída na presença de NaN 3 . A presença de D 2 O aumentou a velocidade de fotooxidação de Trp e BSA e diminuiu a de RBC. Esta diminuição provavelmente está correlacionada com a redução da constante de associação entre InTPP e RBC na presença de D 2 O. Não foi observada variação significativa na fluorescência das biomoléculas ou sobre a porcentagem de hemólise quando supressores de radicais (ferricianeto, manitol e superóxido dismutase) foram usados. Experimentos usando espectroscopia de ressonância paramagnética (EPR) mostraram que somente o O 2 foi gerado por InTPP. Foi proposto um modelo cinético para a fotooxidação de Trp e BSA. A concordância entre os resultados experimentais e este modelo corrobora a predominância do mecanismo via O 2 na fotooxidação das biomoléculas pelo InTPP.


Introduction
Photodynamic therapy (PDT) is a two-step therapeutic modality in which the topical or systemic delivery of photosensitizing drugs is followed by irradiation with visible light, at doses that are not in themselves harmful. 1 For treatment of cancer the photosensitizer is retained preferentially by the tumor and when excited by irradiation generates reactive oxygen species (ROS) that have a cytotoxic effect to the neoplasm. 2These ROS can be generated by two mechanisms, known as type I and type II. 3,4n type I the photosensitizer in the excited triplet state can interact directly with the substrate and/or solvent, through an electron transfer reaction or hydrogen transfer, generating radical ions or neutral radicals, which quickly react with oxygen molecules producing ROS such as superoxide radical (O 2 ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical ( OH), capable of oxidizing a variety of biomolecules.In the type II mechanism, the photosensitizer in the excited triplet state can interact with ground state oxygen molecules ( 3 O 2 ) generating singlet oxygen ( 1 O 2 ) through an energy transfer process. 5Studies in the literature suggest that the photooxidative mechanisms via singlet oxygen are often more efficient than radical processes due to the higher diffusibility of 1 O 2 and the higher reaction rate constants with substrates. 6,7Nonetheless, other studies show that photooxidations occur largely by a type I mechanism. 8,9ome researchers have proposed that the type I mechanism is favored in polar solvents since the radicals generated through electron transfer would be stabilized in solvents with a high dielectric constant. 10,11In contrast, the solubility and the lifetime of singlet oxygen are greater in lipophilic solvents and the type II mechanism is favored in hydrophobic systems.Recently, Vakrat-Haglili et al. 12 showed that the dye's environment determines the mechanism of photooxidation, yield, fate and efficacy of the species involved.Junqueira et al. 13 showed that the negatively charged micelle interface of sodium dodecyl sulfate (SDS) induces dimer formation of methylene blue and shifts a type II to type I mechanism.8][19] Moreover the presence of indium(III) in the core of chloro [13 2 -(dimethoxycarbonyl)pheophorbidato methyl ester]indium (III) enhances in vitro and in vivo photosensitizing efficacies due to the heavy atom effect. 20,21Chloro[13 2 -(dimethoxycarbonyl)pheophorbidato methyl ester]indium (III) encapsulated in liposomes of egg yolk phosphatidylcholine recently entered into phase II clinical trials for ocular photodynamic therapy in choroidal neovascular membranes. 22Recent studies carried out in our laboratory have shown that the singlet oxygen quantum yield of chloro (5,10,15,20tetraphenylporphyrinato)indium(III) (InTPP) (Figure 1) in dimethylsulfoxide (DMSO) ( = 0.72) was higher than that of 5,10,15,20-tetraphenylporphyrin (TPP) ( = 0.52) and that InTPP was an excellent photosensitizer in the photooxidation of tryptophan, bovine albumin and erythrocytes. 23Even with this high value of for InTPP, type I photosensitization pathways cannot be ruled out. 14his work evaluates the main mechanism (type I or II) that acts in the photooxidation of tryptophan, bovine albumin and erythrocytes using InTPP or if both mechanisms proceed simultaneously in the photooxidation of the biomolecules and the cells.For this purpose, the influences of deuterium oxide and singlet oxygen quenchers were studied during the photooxidations.Electron paramagnetic resonance spectroscopy was also used to confirm if 1 O 2 is the main reactive intermediate generated by InTPP for Trp and BSA photooxidation.An explicit mechanistic model for the photooxidation of the biomolecules is proposed based on the elementary steps reported by Rosenkranz et al., 24 considering the experimental results for the main mechanism of photooxidation.

Irradiation source
The irradiation system consisted of a mercury lamp (Phillips® HPLN 80W), a water jacket compartment (which absorbs infrared radiation emitted by the lamp), and a 400/600 nm bandpass filter (Oriel BG 38).The cuvette containing the solutions to be irradiated was placed 5 cm from the source lamp.The irradiance emitted by the mercury lamp, between 400 to 600 nm, was measured with a spectroradiometer (LI-1800, LI-Cor).The value measured was 62.7 W m -2 .All experiments were carried out in a dark room to prevent the influence of surrounding radiation.
The value of the irradiance absorbed by the photosensitizer solutions (I photo, ), expressed as mol photons × m -2 × s -1 at each frequency, was calculated by equation 1: (1)   where I o, is the irradiance emitted by the mercury lamp at each frequency, A is the absorbance of InTPP, h is Planck's constant and N A is Avogadro's constant.The total irradiance absorbed by the photosensitizer (I abs ) was calculated from: I abs = i I photo, .To convert I abs to mol photons × m -3 × s -1 the value of I abs was multiplied by width of the cuvette (10 -2 m).

Fluorescence measurements
Trp and BSA solutions were, respectively, excited at 281 and 279 nm, and fluorescence intensities were monitored at 357 and 326 nm with an ISS PC1 TM -Photon Counting spectrofluorimeter from ISS (Champaign, IL, USA).

Evaluation of the mechanism involved in the photooxidation experiments Trp and BSA photooxidation
Basically, the following photochemical reaction was utilized to determine photooxidation rate constants (k p ): The overall reaction is pseudo first-order with respect to biomolecules because the flux of photons and the concentration of photosensitizer are constant.Considering the concentration of the biomolecules to be proportional to their fluorescence intensity, the following expression is obtained: (3)   where F is the fluorescence intensity of the biomolecules at time t, and F o is the initial fluorescence before irradiation.Graphs of ln(F/F o ) versus irradiation time allow the determination of k p .

Hemolysis of RBC
Photohemolysis was carried out as described by Silva et al. 23 Typically, human blood of a single donor was collected 48 hours before the hemolysis assays in a tube containing EDTA, which was used as an anticoagulant.The tube was kept in a freezer until the moment of use.The serum was separated from the erythrocytes by centrifugation at 780 × g for 10 min.The RBC were washed with NaCl solution (0.85%, m/v) using three times the blood volume and then the cells were centrifuged at 1760 × g for 10 min to reduce the anticoagulant and serum residues.This procedure was repeated three times.RBC solutions (4 mL, 1.89 10 10 cells L −1 ) containing PBS (pH 7.4), Tween 20 (0.45 mmol L −1 ) and DMF (1.6%, v/v) were incubated in the dark with InTPP (6.4 mol L −1 ) for 30 min and then irradiated for 60 min using the photooxidation system previously described.InTPP was initially solubilized in DMF and later added to the solution containing PBS, Tween® 20 and DMF.The involvement of type II and I mechanisms in photohemolysis was evaluated, respectively, by effects of D 2 O (25-75%, v/v) or NaN 3 (50-150 mol L -1 ) and by the influence of radical quenchers (potassium ferricyanide (50-2800 mol L -1 ), mannitol (20-2800 mol L -1 ) or dismutase superoxide (12.5-100.0g mL -1 )).Samples of 550 L were collected at intervals of 10 min and centrifuged at 500 × g for 5 min.The supernatant was analyzed using a Hewlett Packard 8453A Diode Array Spectrophotometer at a wavelength of 542 nm to measure the oxyhemoglobin chromophore released from the erythrocytes as a result of the destruction of the RBC by the photodynamic action of the photosensitizer.The percentage of hemolysis achieved with the photodynamic action was calculated by equation 4: where A 1 is the absorbance of the supernatant from the solution that contains the photosensitizer, A 2 is the absorbance of the control (without photosensitizer), and A T is the absorbance of total hemolysis, which is calculated from the lysis of the erythrocytes with an ultrasound cell disrupter (Ney ULTRAsonik ultrasonic system).

Binding of sensitizers to erythrocytes
The binding constant of sensitizer to erythrocytes in the presence of D 2 O was measured as described by Silva et al. 23 Typically, 1.0 mL of RBC at a total concentration ([P] total ) of 1.1 10 11 cells L −1 in a solution containing 75% D 2 O, PBS (pH 7.4), Tween 20 (0.45 mmol L −1 ) and DMF (1.6%, v/v), were incubated in the dark in the presence of photosensitizer concentrations ranging from 1.0 to 8.0 mol L -1 for 30 min and then centrifuged at 500 × g for 5 min.The concentration of the free photosensitizer was determined spectrofluorimetrically ( excitation = 436 nm and emission = 614 nm), based on a previously constructed calibration curve.Silva et al. 23 showed that the InTPP binding to erythrocytes is cooperative (there are four cooperative binding sites per cell).Then, we calculated the binding constant (K) of InTPP using the equation 5:

EPR measurements
According to Lion et al., 26 the presence of singlet oxygen -in any system -can be confirmed by the detection of a triplet EPR signal assigned to the stable nitroxide radical 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TEMPO).TEMPO is generated by the reaction of TEMP with 1 O 2 (Scheme 1). 26xygen-saturated solutions of TEMP (38 mmol L -1 ) were irradiated for 30 min at room temperature in the presence or absence of InTPP (20 µmol L -1 ), in phosphate buffer (pH 7.2) containing Tween® 20 (8.9 mmol L -1 ) and DMF (5%, v/v).The irradiation system was the same described earlier.EPR spectra were recorded with an ELEXSYS-CW Bruker spectrometer using a rectangular cavity operating in TE 102 mode.Aliquots from irradiated solutions were promptly transferred to a flat quartz cell and measurements were carried out at room temperature with the following instrument settings: microwave power, 20 mW; microwave frequency, 9.685 GHz; field modulation frequency, 100 kHz; field modulation amplitude, 1 G; time constant, 21 ms; and scan time, 84 s.

Oxygen concentration measurements
The oxygen concentrations in the N 2 -and O 2 -saturated phosphate buffer solutions containing InTPP, Trp or BSA, plus Tween® 20 and DMF were measured before the photooxidation experiments using a Clark-type oxygen electrode connected to a computer-operated Oxygraph control unit (Hansatech Instruments, Norfolk, England) at 25 o C.

Determination of the mechanism (type I or II) for biomolecule photooxidation
Photooxidations were carried out under both aerobic and anaerobic conditions to evaluate the involvement of molecular oxygen in the degradation of Trp and BSA.Figures 2a and 2b show a significant reduction of fluorescence intensities (> 70%) for irradiated solutions of Trp and BSA in the presence of O 2 .In contrast, photodegradation was negligible under anaerobiosis (reduction < 34%).These results clearly show that oxygen is essential for the photooxidations.The small amount of degraded Trp and BSA, under anaerobic conditions, probably occurred due to the incomplete removal of oxygen from the solutions.Ericson et al. 27 reported that oxygen is not completely removed from solutions deoxygenated with N 2 .Measurement of the oxygen concentration in the solutions used in these experiments shows that, in anaerobic conditions, the oxygen concentration (139.8 ± 0.8 µmol L -1 ) was 5.2 times smaller than the oxygen concentration measured in aerobic conditions (723.7 ± 0.6 µmol L -1 ).
Figures 3a and 3b show that increasing the concentration of D 2 O to 50%, photooxidation rate constants for Trp and BSA increase 3.9 and 13.9 times, respectively.It is known that the lifetime of singlet oxygen is approximately one order of magnitude higher in D 2 O than in H 2 O. Thus, these results suggest that the biomolecules are photooxidized by a type II mechanism.Conversely, in the presence of 2.0 and 2.6 mmol L -1 sodium azide, rate constants for Trp and BSA decrease, respectively, 4.2 and 3.3 times (Figures 4a  and 4b).This decrease was higher at low concentrations of sodium azide, and lower at higher concentrations.It is known that azide quenches singlet oxygen by two processes (reversible and irreversible). 28The latter is characterized by generation of superoxide and azide radicals which can oxidize biomolecules.Probably at high concentrations of azide ( 0.2 mmol L -1 for Trp and 1.0 mmol L -1 for BSA photooxidations), the generation of radical species accounted for the slower decrease in the rate constants.Otherwise, when a low concentration of a quencher was used, a nonsignificant amount of radical species was generated and an abrupt decrease of rate constant was observed due to the quenching of singlet oxygen.These results are similar to data reported in other research and provide strong evidence to support the hypothesis that the photooxidation of Trp and BSA occurs by a type II mechanism. 10,29,30However, these trials do not allow evaluation of the participation of other active oxygen intermediates during the photooxidation.5][16] Thus, we decided to evaluate the influence of known ROS scavengers on the InTPPsensitized photooxidation of Trp and BSA.Photooxidations were carried out in the presence either of potassium ferricyanide (electron-scavenger), mannitol ( OH quencher) or dismutase superoxide (O 2 suppressor).No alterations in the decay of the biomolecule fluorescence intensities were observed (results not shown).These results show that Trp and BSA are not oxidized by electron transfer, or by hydroxyl or superoxide radicals.
EPR experiments using TEMP as a singlet oxygen trapping agent were carried out to confirm if 1 O 2 is the main reactive intermediate generated by InTPP for biomolecules photooxidation. 26,31The reaction of TEMP with singlet oxygen produces a stable nitroxide radical (TEMPO) (Scheme 1) readily detectable by EPR.Irradiation of an oxygen-saturated phosphate buffer (pH 7.2) solution containing InTPP (20 mol L -1 ) and TEMP (38 mmol L -1 ) led to the generation of a triplet EPR signal (Figure 5a) characteristic of a nitroxide radical (TEMPO in this case). 16,26The intensity of the signal increased rapidly during irradiation of the solution containing InTPP (Figure 5a).In the absence of the photosensitizer, no increase of the signal was observed (Figure 5b).A small triplet signal was present before the irradiation of TEMP due to impurities of TEMPO.In conclusion, these results show that the photooxidation of TEMP proceeds via a type II mechanism, confirming that Trp and BSA are also oxidized by a type II process.

Determination of the mechanism (type I or II) for the RBC photooxidation
Figure 6a shows the results of photohemolysis experiments carried out in phosphate-buffered saline with increasing D 2 O concentrations.There was a marked decrease in the rate of photohemolysis when the concentration of D 2 O is increased from 25 to 75%, v/v.For example, the irradiation time to inactivate 50% of the cell population increased from 29 to 34 min when the D 2 O concentration increased from 0 to 25%, and to 44 and 51 min when 50 and 75% of D 2 O were used.The same situation was observed by De Polis et al. 32 If the photohemolysis proceeds by a singletoxygen mechanism, it would be expected to be faster in D 2 O than in H 2 O because the lifetime of singlet oxygen is greater in the former solvent. 33However, the rate decrease observed should not be taken as an argument against a singlet-oxygen mechanism.As discussed by Valenzeno et al. 34 the D 2 O effects on photohemolysis using other sensitizers are somewhat variable, ranging from no effect to an enhancement of over 200%.We have shown recently that the binding constant between RBC and InTPP is (2.40 0.05) 10 7 L mol -1 , in aqueous solutions. 23  Therefore, InTPP has a high affinity for the erythrocyte membrane due to its lipophilicity. 23It is thus expected that the InTPP molecules enter into the hydrophobic region of the lipid bilayer together with oxygen molecules where the singlet oxygen will be generated.However, the presence of D 2 O in the photooxidation medium decreased the InTPP affinity for the erythrocytes membrane.The binding constant between RBC and InTPP was decreased to (0.92 0.06) 10 7 L mol -1 when 75% D 2 O was used.
Rosen and Klebanoff reported that D 2 O may influence photochemical reactions by exchanging a deuterium for hydrogen in a C-H or an O-H bond that is involved in the reaction. 35The D 2 O may alter the observed reaction rate if a C-D or an O-D bond is involved in the rate-limiting reaction step.Besides, the exchange of deuterium for hydrogen at the binding sites can reduce the affinity of InTPP to bind to RBC.This smaller affinity probably contributed to decrease the photohemolysis in the D 2 O solvent.In this situation, the increase of the D 2 O concentration in the photohemolysis medium decreases the rate of the hemolysis and increases the time needed to inactive the RBC.The effect of sodium azide as a singlet oxygen quencher was also investigated in the photohemolysis.Figure 6b shows that the presence of NaN 3 in the photohemolysis medium affected the ability of InTPP to inactive the RBC.For example, after 60 min of irradiation the average hemolysis percentage of erythrocytes was reduced from 83 to 66% when the azide concentration was increased from 0 to 50 µmol L -1 , and to 55 and 33% for 100 and 150 µmol L -1 of azide.These results suggest that the RBC are photooxidized by a type II mechanism.
Erythrocyte photohemolysis was also carried out in the presence of radical quenchers (potassium ferricyanide, mannitol or dismutase superoxide).No alterations in the hemolysis percentage were observed (results not shown).These results show that erythrocytes are not oxidized by electron transfer, or by hydroxyl or superoxide radicals.

Comparing the photooxidation of BSA and RBC by InTPP
The rate constant for the pseudo first-order photooxidation of the BSA using 6.4 mol L -1 of InTPP is k p = (2.0 0.15) x 10 -4 s -1 .This result was obtained from a graph of ln(F/F o ) versus irradiation time (graph not shown).The half-life of BSA calculated from k p is (57.8 3.5) min.The time to inactivate 50% of the cell population of RBC, obtained with 0% D 2 O (Figure 6a), is (29.2 1.9) min.The association constant for BSA is (1.15 0.07) 10 5 L mol -1 and the binding sites are independent. 23For erythrocytes, the association constant is (2.40 0.05) 10 7 L mol −1 and there are four cooperative binding sites per cell. 23These results suggest that the higher photodynamic activity of InTPP with RBC may be associated with a higher affinity of InTPP for RBC than for BSA.

Kinetic model
As the photooxidation of Trp and BSA occurred via a singlet oxygen-mediated mechanism, we propose a mechanistic model consisting of ten elementary reactions (Table 1) for biomolecule photooxidation mediated by InTPP.The kinetic steps of this mechanism are similar to those proposed by Rosenkranz et al. 24 for the photodynamic inactivation of lysozyme.
P o , 1 P, and 3 P, denote respectively the ground, excited singlet, and excited triplet states of the dye.O 2 , and 1 O 2 , are the ground (triplet) and the excited singlet states of molecular oxygen.S is the substrate (biomolecule) and S-O 2 the product of photooxidation.In their work, Rosenkranz et al. 24 showed that the enzymatic activity of solutions of lysozyme containing acridine orange decreased by an overall pseudo first-order process during irradiation with visible light.Applying quasi-stationary conditions for [ 1 O 2 ] and [ 3 P], the rate constant (k p ) for the process is given by the following expression: 24 where k p is a constant when the sum k 7 + k 8 [S] + k 9 [S] is approximately independent of time. 24This is always the case when k 7 >> k 8 [S] + k 9 [S].T is the triplet quantum yield of the photosensitizer and it represents the relation between the constants k 3 and k 2 ,: T = k 3 /(k 3 +k 2 ). 19Kinetic simulations of the InTPP-sensitized photooxidation of Trp were run with the software Gepasi 3.21. 38,39 The rate constant for step (2), k 2 , was calculated using the experimental values of k f , k isc and f (fluorescence quantum yield) for InTPP. 36The following equations were used: k 2 = k s -k isc , k 3 = k isc and k s = k f / f . 19The constant k 4 was obtained from our own measurement of the triplet lifetime. 23e assumed that singlet oxygen was formed exclusively by energy transfer from the triplet state of the photosensitizer to ground state molecular oxygen.The proportion of 3 P quenched by O 2 is approximately equal to one because k 5 + k 6 >> k 10 .Quenching of 1 P by O 2 was not considered because of the very short lifetime of the first excited singlet state of InTPP. 36Then, k 5 was calculated using the following equations: where is the quantum yield of singlet oxygen.According to the literature, values for k 6 are found in the range 1-3 × 10 9 L mol -1 s -1 , therefore, we assumed k 6 = 1 × 10 9 L mol -1 s -1 . 5Singlet oxygen quantum yields ( = 0.72) were obtained by laser flash photolysis experiments using pheophorbide-a as a reference. 23An oxygraph apparatus was used to determine the concentration of oxygen ([O 2 ] = 723.7 0.6 µmol L -1 ) as described earlier.
The remaining constants in Table 1 were obtained from the literature. 24,37mparing results with the kinetic model where ------------------× -------------- Therefore, a linear relation between k p and [P o ] must be maintained if photooxidations occur by the mechanism suggested in Table 1.For the photooxidation of Trp, experimental k p values increase linearly with [InTPP] (Figure 7b).The same results were observed for the photooxidation of BSA (data not shown).Theoretical values of k p (for Trp) were determined by kinetic simulations (using the software Gepasi 3.21) and plotted for comparison with the experimental data (Figure 7b).The theoretical values are in good agreement with experimental data, validating the photooxidative mechanistic model consisting of ten elementary reactions (Table 1, where 1 O 2 , in step (8), is the only species responsible for the consumption of Trp).Therefore, kinetic modeling also confirms that InTPP photosensitized oxidation of Trp occurs in accordance with a type II process and Trp or BSA photooxidation occurs by the same elementary steps proposed by Rosenkranz et al. 24 for the photodynamic inactivation of lysozyme.To our knowledge, this is the first work showing that the photooxidation of biomolecules by photosensitizer may occur by the same elementary steps as for the inactivation of lysozyme.

Conclusions
In this work, we showed that a type II photodynamic mechanism explains the InTPP-mediated photooxidation of Trp and BSA, because the photooxidation rate constants (k p ) increased in the presence of deuterium oxide and decreased in the presence of sodium azide.The same mechanism also explains red blood cells photooxidation since the hemolysis percentage was reduced in the presence of sodium azide.Evidence in favor of a type II mechanism was also found when potassium ferricyanide, mannitol or dismutase superoxide were used as radical suppressors, because no variations on k p values and percent hemolysis were observed.EPR studies corroborated the proposal that singlet oxygen was the principal oxidant generated by InTPP during the photochemical reactions, due to the detection of a triplet EPR signal assigned to TEMPO.Finally, kinetic simulations for the photooxidation of Trp were performed taking into account a series of 10 proposed elementary reactions (Table 1).Theoretical k p values obtained from the simulations were in good agreement with experimental data, confirming, once more, that photooxidations mediated by InTPP occur by a type II mechanism.
) 1+K[L] n where = [L] bound /[P] total = [sites occupied]/[sites total], n is the number of cooperative sites and [L] is the free concentration of the InTPP.[L] bound in equation 5 was calculated by: [L] bound = [L] total -[L].The parameters n and K of equation 5 were evaluated using the software Origin® 7.5 (OriginLab Inc, Northampton, MA, USA).

Figure 2 .
Figure 2. Evaluation of the involvement of molecular oxygen in the photooxidation of (a) Trp and (b) BSA.N 2 -or O 2 -saturated phosphate buffer solutions (pH 7.2) containing InTPP (15 mol L -1 ), and Trp (150 mol L -1 ) or BSA (150 mol L -1 ) plus Tween 20 (8.9 mmol L -1 ) and DMF (5%, v/v) were irradiated for several min.Fluorescence intensities of the solutions were measured at different times.Data represents mean SD of three independent experiments.

Figure 3 .
Figure 3. Influence of D 2 O on the photooxidation rate constants of (a) Trp and (b) BSA.Oxygen-saturated phosphate buffer solutions (pH 7.2) containing InTPP (20 mol L -1 in (a) and 1.1 mol L -1 in (b)), Trp (150 mol L -1 ) or BSA (23 mol L -1 ) and different concentrations of D 2 O (0-50%, v/v) plus Tween® 20 (8.9 mmol L -1 ) and DMF (5%, v/v) were irradiated for 30-60 min.Fluorescence intensities were measured at different times and k p values were obtained from graphs of ln(F/F o ) versus irradiation time.Data represents mean SD of three independent experiments.

Figure 6 .
Figure 6.Influence of (a) D 2 O and (b) NaN 3 on the photohemolysis of human red blood cells.RBC solutions (1.89 10 10 cells L −1 ) in PBS (pH 7.4) containing InTPP (6.4 mol L −1 ), Tween 20 (0.45 mmol L −1 ), DMF (1.6%, v/v) and different concentrations of D 2 O (25-75%, v/v) or NaN 3 (50-150 mol L -1 ) were irradiated for 60 min.The absorbance of the oxyhemoglobin chromophore released from the erythrocytes as a result of the destruction of the RBC was measured at different times and the percent hemolysis values were obtained from equation (4).Data represents mean SD of three independent experiments.
Figure 7a shows a linear relation between ln(F/F o ) and the irradiation time for Trp in the presence of different concentrations of InTPP, which indicates that the sum k 7 + k 8 [S] + k 9 [S] is approximately independent of time, probably due to the fact that k 7 >> k 8 [S] + k 9 [S].The substitution of I abs for k 1 [P o ] in equation 1 provides a modified expression for k p :

Table 1 .
Elementary reactions of the mechanistic model for biomolecule photooxidation mediated by InTPP I abs calculated for the InTPP concentration of 20 mol L -1 .Vol.19, No. 7, 2008 *