Formation of nickel(II)porphyrin and its interaction with DNA in aqueous medium

In this work, kinetics of the reaction between 5,10,15,20-tetrakis(N-methylpyridium-4-yl)porphyrin and Ni2+ species were investigated in aqueous solution at 25 ±1 oC in I = 0.10 M (NaNO3). Speciation of Ni2+ was carried out in I = 0.10 M (NaNO3) in order to provide the distribution of the Ni2+ species with different solution pH. The experimental data have been compared with the speciation diagram constructed from the values of hydrolysis constants of Ni2+ ion. Speciation data showed that the hexaaquanickel(II), [Ni(H2O)6]2+, ions take place in hydrolysis reactions through formation of [Ni(OH2)6-n(OH)n]2-n species with solution pH. Based on the speciation of Ni2+ and pH dependent rate constants, rate expression can be written as: d[Ni(TMPyP)4+]/dt = (k1[Ni2+(aq)] + k2[Ni(OH)+(aq)] + k3[Ni(OH)2o(aq)] + k4[Ni(OH)3-(aq)])[H2TMPyP4+], where k1, k2, k3 and k4 were found to be k1 = (0.62 ± 0.22) × 10-2; k2 = (3.60 ± 0.40) × 10-2; k3 = (2.09 ± 0.52) × 10-2, k4 = (0.53 ± 0.04) × 10-2 M-1s-1 at 25 ±1 °C, respectively. Kinetic results showed that monohydroxo, [Ni(H2O)5(OH)]+, is the most reactive among the [Ni(OH2)6-n(OH)n]2-n species. The enhanced reactivity has been ascribed to the formation of hydrogen bonding between oxygen atom of hydroxyl group of the [Ni(H2O)5(OH)]+ species and the pyrrolic hydrogen atom of the [H2TMPyP]4+. The rate of formation of [Ni(II)TMPyP]4+ complex was to be 3.99 × 10-2 M-1s-1 in I = 0.10 M, NaNO3 (25 ± 1 oC). Ionic strength effect on the reaction rate is suggested that the net charge of the tetracationic porphyrin is to be +3.6 on the basis of Brønsted-Bjerrum equation. The UV-Vis and fluorescence data revealed that [Ni(II)TMPyP]4+ and H2(TMPyP)4+ interact with DNA, and UV-Vis results suggest that Ni(II)-porphyrin and free base porphyrin interact with DNA via outside binding with self-stacking and intercalation, respectively. Mechanism of kinetics of formation of the [Ni(II)TMPyP]4+ complex in aqueous medium is discussed. An investigation of application of the [Ni(II)TMPyP]4+ complex along with other metalloporphyrins such as Zn2+-, Ru2+-, Pt2+-, [Au(III)TMPyP]5+ as anti-COVID-19 agents is now in progress under international collaboration.

In the human body system, the protoporphyrin IX ring is continuously synthesized during biosynthesis of heme, and iron(II) is subsequently coordinated to the porphyrin core. Studies of kinetics of incorporation of metal ions into the porphyrins' core give the mechanistic pathways of the formation of metalloporphyrins. By knowing proper reaction pathways of formation of metalloporphyrins, it may possible to formulate porphyrin-based new drugs. Hambright and Chock (1974) proposed a general mechanism of formation of metalloporphyrins for the first time, and later that was reviewed by a number of research groups [18][19][20][21][22][23][24][25]. Open chain-ligands exhibit significant reactivity in formation of metallocomplexes while macrocyclic ligands, for example, porphyrins, show poor reactivity because of their complex nature [26]. Several approaches have already been executed to accelerate the metalation reaction of the porphyrins. Some of the events should be approaches: (i) substitution reactions of cadmium(II) or mercury(II)porphyrins [27][28][29], (ii) porphyrins with substituents at the pyrrole nitrogen [30,31], (iii) functionalization of porphyrins (e.g., tetracarboxylic acid "pocket-fence" porphyrins) (iv) performing reactions at a suitable solution pH in presence of metal ions having hydroxo-ligands [20,24,25]. The rate of formation of metalloporphyrins could be enhanced via any of the above-mentioned approaches. Accordingly, a group of researchers showed the enhanced reactivity of macrocyclic porphyrins for the metal ions having hydroxo-ligands [Tanaka, 1983;Habib et al., 2020]. They reported that the presence of hydroxo-ligands of the metal ions facilitates the formation of hydrogen bonding with the pyrrolic hydrogen atom of the free base porphyrin [20,24,25]. Batinić-Haberle et al. (1999) also studied the metalation reactions of incorporation of Fe 3+ and Mn 3+ into the 5,10,15,20-tetrakis(N-alkylpyridiniumyl)porphyrins and they found the enhanced reactivity for the exchange metalation of monohydroxoiron with the aquamanganese porphyrins because of trans-axial effect [4].
Nickel is an essential element for humans as well as for other animals in functioning many metabolic reactions. The known multifunctional properties of the porphyrinson o and metalloporphyrins have been extended the porphyrins' research in various fields. So, the study of kinetics and mechanism of the formation of Ni(II)porphyrin may open a new research arena of applications of nickel-porphyrin complexes. Reduced form of the nickel-porphyrin complexes acts as enzymatic cofactors (F430) in the global carbon cycle and also used in solar-fuel cells as a powerful catalyst to evolve the hydrogen gas. The reduced nickel-porphyrins have been used in the reduction of CO and CO2 as well. Nickel is a transition metal having d 8 electronic configuration, thus exhibits least reactivity in complex formation. However, in our previous study, we found enhanced reactivity of Au 3+ ion in the formation of complexes with the macrocyclic tetrakis(N-methylpyridinium-4-yl)porphyrin, [H2TMPyP] 4+ , where Au 3+ ion belongs to the d 8 electronic configuration [24]. According to the speciation diagram of Au 3+ ion with solution pH, the monohydroxotrichloroaurate(III), [AuCl3(OH)] -, was found as a predominant species under the experimental condition [4]. The negatively charged [AuCl3(OH)] -ion can easily approach the core of the tetracationic porphyrin and the presence of the hydroxo-ligand in the Au 3+ species causes enhanced reactivity in the formation of the [Au(III)TMPyP] 5+ complex. This is because the hydroxo-ligand of the [AuCl3(OH)] -species forms hydrogen bonding with the pyrrolic hydrogen atom of the porphyrin, which resulted in enhanced rate of the reaction. A number of research groups has also attempted to investigate the kinetics of formation of Ni(II)porphyrins to understand the mechanism of reaction [32][33][34][35]. Tan et al. (2011) and Tian et al. (2012) studied the speciation of nickel from the geo-and hydrothermal points of view, however, limited information is available to understand the kinetics of the reaction of Ni 2+ with H2TMPyP 4+ [36,37].
Very recently Liu and Li (2020) studied the severe health effect by the novel coronavirus (COVID-19) worldwide by applying theoretical models [52]. They used conserved domain analysis, homology modeling and molecular docking models to compare the biological roles of specific proteins of the COVID-19, and found the novel coronavirus attacks the 1-beta chain of the hemoglobin and captures the protoporphyrin IX to inhibit human heme metabolism. The theoretical results suggest that the COVID-19 has strong affinity for the porphyrins and/or metalloporphyrins. The noble but clinically relevant finding encouraged us for investigation of possible applications of the porphyrins as anti-COVID-19 agents.
In this paper, speciation of Ni 2+ in aqueous medium with different solution pH in I = 0.10 M (NaNO3) and 0.10 M NaCl at 25 ±1 °C has been characterized. By applying the distribution of the Ni 2+ species with solution pH, the kinetics of the formation of [Ni(II)TMPyP] 4+ complex, I (Scheme 1) has been studied in order to explore the reactions mechanism for the metalation reaction. We have extended our studies on interaction of DNA with the [Ni(II)TMPyP] 4+ complex along the H2TMPyP 4+ in order to investigate their potential applications in the medical as well as in the biological fields. An investigation of the application of porphyrins, particularly Ni 2+ -, Zn 2+ -, Ru 2+ -, Pt 2+ -, [Au(III)TMPyP] 5+ as anti-COVID-19 agents is now in progress under international research collaboration. Scheme 1. Tetracationic nickel(II)porphyrin, I.

Results and discussion
3.1 Speciation of Ni 2+ Speciation of Ni 2+ ion in aqueous solution in the presence of 0.10 M of NaNO3 (I) at 25 ±1 °C was carried out to investigate the kinetics of the reaction between the free base porphyrin, [H2TMPyP] 4+ , and the Ni 2+ species. In order to investigate the kinetics of the metalation reaction, it is highly expected to know the speciation of the relevant metal ion. This is because the speciation diagram provides species distribution that is required to establish the reaction mechanism for the relevant reaction. Figure 1 shows the speciation diagram generated from the hydrolysis constants of Ni 2+ species with the solution pH [53]. As seen from Fig. 1 4+ , because of its very poor existence in the aqueous system under the present experimental conditions. The solubility product for the dihydroxo Ni 2+ species is only K b S10 = -15.7, thus it starts to precipitate at pH ~8.15 for 10-3 M of Ni 2+ solution [53]. This causes the presence of a small fraction of the [Ni(H2O)4(OH)2] 0 species in this study ( Fig. 1). According to the speciation diagram, the [Ni(H2O)4(OH)2] 0 species is distributed from pH ~8.25 to the higher pH ( ≥ 12.00) where its maximum distribution is observed at pH ~10.30 ( Fig. 1). It is expected that the reactivity for the dihydroxo Ni 2+ , [Ni(H2O)4(OH)2] 0 , species towards the free base porphyrin, [H2TMPyP] 4+ , would be higher than that of the monohydroxo, [Ni(H2O)5(OH)] + , species. This is because the dihydroxo species is electrically neutral, thus the [Ni(H2O)4(OH)2] 0 species can easily approach to the tetracationic porphyrin's core without any Coulombic force of repulsion while the unipositive aqua monohydroxo Ni 2+ , [Ni(H2O)5(OH)] + , species could suffer from repulsive force. The dihydroxo Ni 2+ species exists with a small fraction in the aqueous systems because of its very low solubility product, K b S10 = -15.7, under the present experimental conditions ([Ni 2+ ] = 10 -3 M; I = 0.10 M, NaNO3), therefore, it is reasonable to observe the less reactivity for the [Ni(H2O)4(OH)2] 0 species towards the [H2TMPyP] 4+ as described in the kinetics section. The trihydroxo Ni 2+ , [Ni(H2O)3(OH)3]species is distributed from pH 9.4 to the higher pH > 12.00 while its distribution is so small ~1-2% at the experimental solution pH, 9.50 (Fig. 1).
The UV-Vis spectral data also confirm the stepwise formation of the hydroxo Ni 2+ , [Ni(H2O)6-n(OH)n] 2-n , species as a function of the solution pH (supplementary Figure S-1). As mentioned above, the Ni 2+ exists in aqueous system as hexaaqua, [Ni(H2O)6] 2+ , species. The [Ni(H2O)6] 2+ species shows the ligand to metal charge transfer (LMCT) transition and the absorption maximum is centered at λmax = 391 nm in the UV region (supplementary Figure S-1). The LMCT transitions have been assigned due to the charge transfer from bonding or nonbonding p-orbital of ligand to high energy antibonding dp*-orbital of the metal ion [54]. The UV-Vis absorption spectra for Ni 2+ (1.  Fig. 7. The variation of the absorbance depicted as ln(At-Aα) was plotted with time in order to achieve the observed rate constants for the reactions between the [H2TMPyP] 4+ and [Ni(H2O)6-n(OH)n] 2-n species as a function of solution pH.
The rate of formation of the [Ni(II)TMPyP] 4+ complex is first order with respect to the free base porphyrin that can be written by the following equation: where kobs is the observed first-order rate constant and kf is the second order formation rate constant.
From the reactions between the [H2TMPyP] 4+ and Ni 2+ species at different solution pH, the observed rate constants (kobs) were measured to explore the reactivity of the various [Ni(H2O)6-n(OH)n]2-n species. The values of the observed rate constants for the reactions of the free base porphyrins with the Ni 2+ species as a function of solution pH are shown in Fig. 2. As seen from Figure 2, the observed rate constant increases as a function of the solution pH and goes to its maximum value at pH 9.50 and then slow down as pH is being increased. The rising trend for the rate constants almost remains constant until pH 6.60 and then increases sharply with pH. These results suggest that the reacting species of the Ni 2+ ion is mostly hexaaqua Ni 2+ ion, [Ni(H2O)6] 2+ written as Ni 2+ (aq), within the pH range from 2.97 to ~8.00 which is one of the less reactive among the [Ni(H2O)6-n(OH)n] 2-n [n = 1, ….,6] species (Fig. 1). ] species as a function of solution pH that stated in equations 1-4. In our previous study, it has also been reported that Zn 2+ ion exists predominantly as a hexaaqua, [Zn(H2O)6] 2+ , species at low pH (~2-5) in 0.10 M NaNO3, and changes to hydroxo species stepwise and finally converts to tetrahydroxo [Zn(OH)4 2-] species at higher solution pH [25].
It is noteworthy to mention that the OHgroup coordinated to the metal ion plays crucial role for enhancing the reactivity of the metal species towards the [H2TMPyP] 4+ . This is because the oxygen atom of the aqua-monohydroxo, [Ni(H2O)5(OH)] + , species forms hydrogen bonding with the pyrrolic hydrogen atom of the free base porphyrin, thus the aqua-monohydroxo species can easily approach to the porphyrin's core. The easy approach of the aqua-monohydroxonickel(II) species enhances its reactivity. On the other hand, the aqua Ni 2+ , [Ni(H2O)6] 2+ , species lacks of hydroxo ligand, so the aqua-species is incapable to form hydrogen bonding with the pyrrolic hydrogen atom. This is the reason for its less and/or least reactivity towards the [H2TMPyP] 4+ . However, the aqua-dihydroxo, [Ni(H2O)4(OH)2] 0 , species exhibited less reactivity with the free base porphyrin, [H2TMPyP] 4+ . This is because the aqua-dihydroxonickel(II) species takes part in hydrolysis reaction at solution pH 8.20 and then phases out from the aqueous system through precipitation reaction at higher pH, e.g., 9.50. The precipitation reaction causes lesser distribution of the dihydroxonickel(II) species compared to the aqua-monohydroxonickel(II) species at solution pH 9.50 (Fig. 1). The anionic trihydroxonickelate(II), [Ni(H2O)3(OH)3] -, species seems to be exhibited better reactivity towards the cationic porphyrin ([H2TMPyP] 4+ ), however, the presence of the higher number of the hydroxo groups slows down its kinetics [24, 25,30,55]. These results suggest that though the first OH-ligand is responsible for the formation of hydrogen bonding with the pyrrolic hydrogen atom, however, displacement of the remaining OHseems slow. It has been reported that the OHgroup is strongly coordinated to the metal ion having d 8 electronic configuration like Pt 2+ ion [59]. The electronic configuration of Ni 2+ is also d 8 , so their chemical properties are supposed to be similar; hence the OHgroups are also strongly coordinated with the Ni 2+ ion. Bailey and Hambright (2003) reported that Cu 2+ ion exhibited the highest reactivity among the other first transition metal ions, such as Zn 2+ , Co 2+ and Ni 2+ towards the free base H2-BrP(4) 4+ and tricationic H-BrP(4) 3+ porphyrins at 25 °C in I = 0.10 M (NaNO3) and the reactivity order was found to be Cu 2+ > Zn 2+ > Co 2+ > Ni 2+ [60]. It is expected that the reactivity of Ni 2+ among the divalent metal ions towards the porphyrins would be less because of its d 8 electronic configuration. However, the presence of hydroxo-ligands with the Ni 2+ species enhances its reactivity in incorporation with the free base porphyrin, [H2TMPyP] 4+ . Similar results have also been observed for Au 3+ ion (d 8

Observed rate constants (kobs) as a function of concentration of Ni 2+
Kinetics of the incorporation of Ni 2+ ion into the [H2TMPyP] 4+ with variation of the concentration of Ni 2+ (I = 0.10 M, NaNO3; pH 9.50) at 25 ±1 °C has also been studied in order to ascertain the formation rate constant for the metalation reaction. The observed rate constants (kobs) with concentration of Ni 2+ were obtained by plotting the ln(At-Aα) vs time. The formation rate constant was obtained by plotting the observed rate constants (kobs) with the concentration of Ni 2+ as depicted in Figure 3. As seen from Fig. 3, the rate constant for the metalation reaction increases with increasing the concentration of Ni 2+ and passes through the origin (r 2 = 0.999). This result suggests that the metalation reaction depends on concentrations of the both reacting species and follows the first order kinet-ics. The formation rate constant (kf) for the [Ni(H2O)5(OH)] + /[H2TMPyP] 4+ was found to be 3.99 × 10 -2 M -1 s -1 in I = 0.10 M (NaNO3) at 25 ±1 ºC.

Observed rate constants (kobs) with ionic strength
The rate constants of a reaction for opposite charged reacting species decrease as the ionic strength increases while that increase for the same charged species [61]. Figure 4 shows the dependence the ionic strength on the rate constants for the reaction between the free base porphyrin, [H2TMPyP] 4+ , and Ni 2+ species in I = 0 -10.0 × 10 -2 M (NaNO3) at pH 9.50 where the experimental conditions were kept constant. The observed rate constants (kobs) were obtained by plotting the ln(At-Aα) vs time at different ionic strengths (Fig. 4). As seen from Figure 4, the observed rate constants (kobs) exponentially decrease with the ionic strengths. These results suggest that the reacting compounds exist as oppositely charged species in solution, however, the speciation diagram is indicating the existence of the monopositive monohydroxo Ni 2+ , [Ni(H2O)5(OH)] + , species at solution pH 9.50. In our previous study, we also found the retardation of the kinetics between the dihydroxo Zn 2+ , [Zn(H2O)4(OH)2] 0 , species and [H2TMPyP] 4+ in the presence of NaNO3 (I = 0.10 M) and the net charge of the porphyrin, [H2TMPyP] 4+ , was calculated to be +3.4 [Habib et al., 2020]. The rate constants for the incorporation of monohydroxotrichloroaurate(III), [AuCl3(OH)] -, into the [H2TMPyP] 4+ also decrease exponentially with ionic strength and the calculated net charge of the free base porphyrin was found to be +3.4 by using the Fuoss equation [24]. However, in this study, we found a less decreasing tendency of the rate constants as the ionic strength increases. This may be due to the existence of monopositive monohydroxo Ni 2+ , [Ni(H2O)5(OH)] + , species that form adducts with the anionic/cationic species in the aqueous solution at pH 9.50 ( Fig. 1 and Fig. 4). Hambright (2002) also reported that the rate constants for the reactions of the [H2TMPyP] 4+ with Zn 2+ species decrease as ionic strength increases, and the apparent net charge of the tetracationic porphyrin was found to be +1.4 by using the Bronsted-Bjerrum equation [6]. It seems that the direct reaction of the dipositive Ni 2+ ion with the [H2TMPyP] 4+ is strenuous, thus the presence of the nitrate ions causes to reduce the repulsive force between the [Ni(H2O)5(OH)] + species and the [H2TMPyP] 4+ by interacting with the positively charged tetracationic porphyrin that facilitates the formation of aggregates of cation-anion with lower positive charge. Thus, it is assumed that the tetracationic free base porphyrin, [H2TMPyP] 4+ , carries relatively lower charge than the actual charge, +4.0 and probably both the monopositive monohydroxo, [Ni(H2O)5(OH)] + , and neutral dihydroxo, [Ni(H2O)4(OH)2] 0 , species of Ni 2+ ion took part in the reactions with the free base porphyrin, [H2TMPyP] 4+ , at pH 9.50 [6,24,25].   Figure 5 shows the Brønsted-Bjerrum plot for incorporation of Ni 2+ ion into the tetracationic porphyrin, [H2TMPyP] 4+ . As seen from Fig. 5, regression coefficient (R 2 ), error bars and slope for the plot are 0.974, 1% and -3.60, respectively. It is expected that the intercept for the plot of logkobs vs √I should be zero, however, that is to be -0.273. This is because of the presence of inherent ions that cause intrinsic ionic strength. The slope for the plot is -3.60 that corresponds to ZAZB. Thus, it is expected that the net charge of the tetracationic porphyrin would be +3.6. Nwaeme and Hambright (1984) studied the effects of ionic strength on the rate of the reactions for both the positive and negative porphyrins with divalent metal ions [62]. They reported that the rates of the reactions for positive porphyrins with positive divalent metal ions increase as increasing the ionic strength and that decrease for oppositely charged reacting species with the ionic strength. Williams et al. (1979) also reported the anionic effect on the reaction rate for tetracationic porphyrins in detergent solution [63].
According to the speciation diagram (Fig. 1), the predominant species of Ni 2+ is monopositive monohydroxo, [Ni(H2O)5(OH)] + , at solution pH 9.50. Thus, the rate of reaction of the tetracationic tetrakis(N-methylpyridium-4-yl)porphyrin, [H2TMPyP] 4+ , with the monopositive monohydroxonickel(II), [Ni(H2O)5(OH)] + , species should be increased with increasing the ionic strength, however, that decreases as a function of ionic strength (Fig. 4). These results are suggesting the presence of anionic species of Ni 2+ under the experimental conditions, however, speciation diagram ( Fig. 1) shows the presence of monopositive monohydroxo, [Ni(H2O)5(OH)] + , species at solution pH 9.50. The monopositive Ni 2+ species, [Ni(H2O)5(OH)] + , may associate with the inherent anion, thus forms negatively charged inner sphere reacting species in character such as ([Ni(H2O)5(OH)] + -inherent anion) - [58,64]. The addition of nitrate ion reduces the positive charge of the cationic porphyrin at the transition state through formation of anion-cation-porphyrin adducts that causes the decreasing the reaction rates for negatively charged nickel species [18,58,65].   Figure S-2b). The heavy atom effect by the nickel causes weak intensity for the [Ni(II)TMPyP] 4+ complex. In our previous study, we also found weak intensities from the Ru 2+ -, Pd 2+ -, Pt 2+ -and [Au(III)TMPyP] 5+ porphyrins in aqueous solution because of the heavy atom effect [40,41]. The fluorescence intensity for the [Ni(II)TMPyP] 4+ complex is significantly decreased upon addition of a low concentration of DNA, and then increased with further addition of DNA (supplementary Figure S-2a). The porphyrin molecules aggregate on the negatively charged phosphate network of the DNA molecules through self-stacking in the presence of low concentration of DNA, however, de-aggregation occurs upon addition of additional DNA. This causes the increasing the fluorescence intensity of the metalloporphyrin [40,41]. On the other hand, the intensity of the fluorescence centered at 660 nm did not change but the intensity of the hump appeared at ~628 nm is increased with a low concentration of DNA into the [H2TMPyP] 4+ solution. The intensity of the hump increases with addition of DNA and the fluorescence spectrum is finally centered at 630 nm (supplementary Figure S-2b). These results suggested that the cationic free base porphyrin initially interacts with DNA via negatively charged phosphate network in the presence of a low concentration of DNA, and then de-stacking occurs upon further addition of DNA [40,41]. From the UV-vis and fluorescence spectral results, it is confirmed that both the metalloporphyrin, [Ni(II)TMPyP] 4+ , and the free base porphyrin interact with DNA but their modes of interaction are different. As seen from Figure 6a, the hypochromicity and Bathochromic shift (Δλ) for the [Ni(II)TMPyP] 4+ are only ~ 13% (at λmax 436 nm) and ~1 nm upon addition of high concentration of DNA, respectively. These results suggested that [Ni(II)TMPyP] 4+ interacts with DNA via outside binding with self-stacking [38][39][40][41]50,51]. However, the significant hypochromicity (~ 31% at 422 nm) and a wide Bathochromic shift (Δλ = 17 nm) for the free base porphyrin upon addition of the same amount of DNA confirm its interaction with DNA through intercalation [38][39][40][41]50,51]. The presence of metal ion in the porphyrin core is responsible for carrying water molecules as axial ligands that make the bulkiness of the metalloporphyrin molecules. The large size of the metalloporphyrin molecules interact with DNA via outside binding rather than intercalation, however, the free base porphyrin interacts with DNA via intercalation because of its smaller size that facilitates easy excess into the DNA grooves. Metalloporphyrins that are outside binders have catalytic effects to cleave DNA [40,41,66], thus it is expected that the [Ni(II)TMPyP] 4+ complex can be used as a chemotherapeutic agent in the medical as well as in the biological fields. was prepared by dissolving requisite amount of NiCl2.6H2O (Merck, Germany) in aqueous solution and the concentration was measured by using an atomic absorption spectrophotometer (Perkin Elmer, AAanalyst 200). Sodium nitrate, sodium hydroxide and hydrochloric acid were purchased from Merck, Germany. All the chemicals/reagents were used without further purification. Tetracation nickel(II) porphyrin, [Ni(II)TMPyP] 4+ , was prepared and absorption spectra were recorded in water at pH 9.50 containing 0.10 M NaNO3. Absorption maximum (λmax) and molar extinction coefficient (ε) of the prepared [Ni(II)TMPyP] 4+ complex were 436 nm and 114 × 10 3 M -1 cm -1 , respectively (Fig. 1) [67]. A stock solution of salmon fish sperm DNA, purchased from Sigma-Aldrich, was prepared by dissolving in distilled water and the concentration in base pairs was determined by knowing the absorbance at λmax = 260 nm and using the molar extinction coefficient, ε260 =1.32 × 10 4 M -1 cm -1 [40,41]. Stock solution of the DNA was kept in a refrigerator at -4 ºC. The frozen DNA solution was incubated in a water bath at 37 ºC for an hr and diluted as required before the experiment. Acetate/sodium acetate and 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, Sigma-Aldrich) buffer solution were prepared in 100 mL distilled water as stock solutions and used with required dilution throughout the experiments. pH of the HEPES solution (0.10 M) was adjusted to 7.40 upon addition of either NaOH or HCl. In this work, distilled water was used to perform all the experiments.

Speciation of Ni(II) complexes
Solutions of 5.00 × 10 -3 M NiCl2 with changing solution pH from 2.97 to 11.40, were prepared in 50 mL volumetric flasks separately. The requisite volume of sodium nitrate was added to each solution in order to maintain ionic strength (I = 0.10 M). Solution pH was adjusted by addition of either HCl or NaOH in acetate buffer ([Acetate] = 0.02 M). The UV-Vis spectra of the Ni 2+ species were recorded by using a double beam UV-Vis spectrophotometer (SHIMADZU, Model UV-1800) within a range from 350 to 500 nm. A number of Ni 2+ solutions (5.00 × 10 -3 M) with different concentration of acetate ion ranging from 0 to 1.00 ×10 -2 M was prepared under the same experimental conditions to investigate the interaction between Ni 2+ and acetate ions and found no formation of Ni(II)-acetate complex. A pH meter (HANNA HI 2211) was used to measure the solution pH.

Kinetics of formation of [Ni(II)TMPyP] 4+ complex
Pseudo-first order condition was kept constant throughout the experiment in order to explore the kinetics of the reactions between tetracationic free base porphyrin and Ni 2+ species in I = 0.10 M (NaNO3) at 25 ±1 ºC where the pH of the solutions were varied from 2.97 to 11.05. Concentration of Ni 2+ was varied from 0.50 × 10 -3 to 5.00 × 10 -3 M while that for the porphyrin, [H2TMPyP] 4+ , was kept constant at 1.24 × 10 -5 M. The metalloporphyrin was prepared by mixing the porphyrin solution with the Ni 2+ solution in a 1-cm cell compartment and pre-equilibrated at 25 ±1 ºC. The change in the absorbance was monitored as a function of time at 422 nm (λmax of [H2TMPyP] 4+ ) by using a UV-Vis spectrophotometer (SHIMADZU, Model UV-1800). Formation of the [Ni(II)TMPyP] 4+ complex was monitored by observing isosbestic points at 431, 490 and 546 nm in the visible region as the porphyrin reacted with the Ni 2+ species. Appearing the isosbestic points is confirming the free base porphyrin and Ni(II)porphyrin complex are only the absorbing species. Figure 7 shows such a spectral pattern of the formation of the [Ni(II)TMPyP] 4+ complex with time. To obtain the observed rate constants (kobs), values of ln(At-Aα) were plotted with time and found linearity over two half-lives. Rate constants for the reactions between the free base porphyrin and Ni 2+ species were determined by varying solution pH, nickel concentrations and ionic strengths. The duplicate runs under the same conditions agreed within 5% error. A pH meter (HANNA HI 2211) was used to measure the solutions pH.

Interaction of [Ni(II)TMPyP] 4+ complex with DNA
The UV-Vis spectra of the free base porphyrin and [Ni(II)TMPyP] 4+ complex upon addition of DNA were recorded by using a double-beam UV-Vis spectrophotometer (UV-1800, Shimadzu, Japan). A fluorescence spectrophotometer (F-7000, Hitachi, Japan) was used to record the luminescence spectra for the free base porphyrin and the Ni(II)porphyrin in the presence of DNA. The fluorescence emission wavelength was scanned from 550 to 800 nm by setting the excitation wavelength at 446 and 431 nm for the [Ni(II)TMPyP] 4+ and [H2TMPyP] 4+ , respectively. This is because the isosbestic points for the binary system of [Ni(II)TMPyP] 4+ -DNA and [H2TMPyP] 4+ -DNA were observed at 446 and 431 nm, respectively. Under the present experimental conditions, for 1.14 × 10 -5 M of [Ni(II)TMPyP] 4+ and [H2TMPyP] 4+ , the absorbance and luminescence spectra of the porphyrin solutions were not affected by the species adsorbed on the surface of the cell wall. These were confirmed by recording a UV-vis spectrum of ethanol-water after discarding the analyte solution and found no peaks from the ethanol-water. All the experiments were carried out under room light. HEPES solution of 0.02 M (pH 7.40) was used throughout the experiment. A pH meter (HANNA HI 2211) was used to measure the solution pH.

Conclusion
In this work, kinetics and mechanism of formation of [Ni(II)TMPyP] 4+ have been studied at 25 ±1 ºC in I = 0.10 M (NaNO3) within a pH range from 2.97 to 11.40 in aqueous medium. Speciation of Ni 2+ ions in aqueous medium has also been done in 0.10 M NaNO3 in order to provide the distribution of the Ni 2+ species as a function of solution pH for the kinetic study. The experimental data have been compared with the speciation diagram generated from the values of hydrolysis constants of Ni 2+ ion.