Photo-activated proflavine degrades protein and impairs enzyme activity: Involvement of hydroxyl radicals

Graphical abstract


Introduction
Proflavine, an acridine dye, is commonly used as an antiseptic in wound treatments as well as bacterium disinfection. It was also discovered to be too toxic to be used as a systemic anti-bacterial [1,2]. Numerous publications showed frameshift mutations, when viruses, bacteria, cultured cells, and bacteriophages were treated with proflavine [1][2][3]. Furthermore, multiple investigations have revealed proflavine's mutagenic nature, as well as its significant hazard potential [2,4,5]. Proflavine can enter the epidermal and dermal layers of cells and concentrate in their nucleus [6,7]. This dye has also been discovered to impede cancer cell multiplication by intercalating between DNA base pairs [8,9]. Because of its capacity to interact with DNA double helix, it has the potential to be developed as an anticancer medication [10][11][12]. In vitro cytotoxicity tests on breast and ovarian cancer cell lines revealed that combining the medication with metal ions can result in a more effective antineoplastic agent [13]. When exposed to visible light, proflavine produces numerous types of reactive oxygen species, but only when a macromolecule like DNA is involved in the reaction [14]. Proflavine can generate double-stranded breaks in DNA in the presence of light, as we and others have previously discovered through free radical generation [4,6]. In addition, proflavine produced superoxide ion (O 2 •− ) and caused structural changes in proteins, which was amplified when the divalent metal ion Cu (II) was added [15]. Copper ions are important trace element that is found throughout the body. The liver and kidney have a relatively high concentration of Cu (II), whereas typical serum contains up to 8 μM loosely bound copper that can be used in redox reactions especially in catalyzing the reactive oxygen species (ROS) formation [16,17].
Reactive oxygen species (ROS) accumulation can lead to oxidative stress, which has been linked to the development of a variety of chronic diseases and biological processes such as apoptosis, necrosis, autophagy, and inflammation [18][19][20][21][22][23]. Superoxide anion production from proflavine may cause calf thymus and supercoiled plasmid DNA breakage, as well as protein degradation and red blood cell hemolysis [24,25]. Proflavine can produce (O 2 •− ) through a direct interaction between an electron expelled by excited proflavine and molecular oxygen or through the decomposition of peroxide radical ( • OOH). In the presence of H 2 O and O 2 , photoexcited proflavine can then give birth to 3 O 2 and 1 O 2 via direct energy transfer, as we previously stated can accept an electron from a molecule of oxygen and produce a peroxide radical. In the presence of H 2 O, this peroxide radical can produce hydroxyl radical ( • OH) or • OOH in the presence of copper. These free radicals may assault the target molecules and cause additional damage. DNA and protein damage, oxidation of polyunsaturated fatty acids, oxidation of amino acids, and deactivation of particular enzymes are all harmful effects of ROS, resulting in metabolic and cellular problems [26]. Proteins are the primary targets of ROS, which can play a regulating role in cellular remodeling and proliferation [27,28]. The harmful involvement of oxidized proteins in the etiology of Alzheimer's disease has recently been hypothesized [29]. Furthermore, ROS plays a role in diseases like arthritis, diabetes, cancer, and cardiovascular disease [22,23,[30][31][32][33].
Given the widespread use of proflavine and acriflavine compounds in the treatment of infected wounds, as antimalarial and antiprotozoal agents, and as an anticancer agent, it seemed prudent to conduct toxicity tests on macromolecules, particularly enzymes, in conditions where photoexcitation in the presence of metals could be expected. In this study, we investigated the effect of photo-illuminated proflavine on protein degradation and loss of proteolytic activity of the enzyme trypsin through the generation of hydroxyl radicals, because there is no report in the literature on the deactivation of the enzyme by photoilluminated proflavine. Additionally, it is important to look at how proflavine affects the structure and function of prospective proteolytic enzymes. The findings of this study might contribute to a better understanding of proflavine's negative effects on protein and enzyme breakdown caused by oxygen-free radicals.

Detection of hydroxyl radicals ( • OH)
The production of hydroxyl radicals was quantified using a modified method of Halliwell and Gutteridge's [34]. A 0.5 mL reaction mixture comprising 10 mM sodium phosphate buffer pH 7.4, 2 mM deoxyribose, and various concentrations of proflavine was incubated with 800 lx of cool fluorescent light for five hours at 37 • C. The reaction mixture was then heated at 100 • C for 30 min before being cooled to measure absorbance at 532 nm against a blank that did not contain proflavine. To prevent the production of hydroxyl radicals, hydroxyl radical scavengers such as mannitol, thiourea, and sodium formate were utilized as inhibitors. Experiments were carried out three times, with the mean data being recorded each time.

Proflavine induced BSA degradation and trypsin digestion assay
Schroeder method was used for protein degradation and a trypsin enzymatic assay [35]. A total volume of 1 mL of 10 mM sodium phosphate buffer, pH 7.4, with 2 mg/mL of BSA and different doses of proflavine with or without 200 μM CuCl 2 was used in the reaction mixture. The samples were then treated with 10 μg trypsin for 30 min at 37 • C after being illuminated with fluorescent light. 0.1 mL of 10 mM EDTA and 0.5 mL of 10 % trichloroacetic acid (TCA) were used to stop the reaction. The samples were then centrifuged for 10 min at 2500 rpm to remove the undigested protein (precipitate), and the TCA soluble material in the supernatant was used to determine the acid-soluble peptide using the Lowry et al. [36] method, with the color development being read at 660 nm against a reagent blank. Beckman DU-40 spectrophotometer was used for measuring absorbance.

BSA and trypsin degradation by proflavine and SDS-gel electrophoresis
At different time intervals, the reaction mixture in a total volume of 1 mL 10 mM sodium phosphate buffer pH 7.4 containing either BSA (1 mg/mL) or heat-inactivated trypsin (2 mg/mL) plus proflavine in the presence or absence of 200 μM Cu(II) were incubated with fluorescent light at room temperature. To avoid auto-proteolysis, the trypsin was boiled for 3 min before being used in the procedure. Free radical scavengers such as sodium azide, potassium iodide, sodium benzoate, sodium formate, thiourea, and mannitol were added at a concentration of 50 mM to the protein-proflavine reaction mixture in several tests. The reaction was stopped with 0.025 mL of SDS-sample dye, which consisted of 62.5 mM Tris-HCl pH 6.8, 10 % (v/v) glycerol, 2 % (v/v) β-mercaptoethanol, and 0.001 % (w/v) bromophenol blue. The samples were boiled for five minutes before being loaded with 10 mg of BSA in each well. According to the Laemmli [37] method, the samples were separated on 10 % (w/v) SDS-polyacrylamide gels. In the case of trypsin, 10 μg protein was applied to each lane, and the samples were separated on SDS-polyacrylamide gels containing 15 % (w/v) SDS. After electrophoresis, the gels were stained with silver nitrate staining, as described by Nesterenko et al. [38]. The TCA soluble product was measured after BSA was treated with 10 μg of trypsin for 30 min at 37 • C.

Spectroscopy
Fluorescence spectra were generated using a Shimadzu spectrofluorometer (Tokyo, Japan). 0.7 μM of BSA and lysozyme were stimulated (λ max ) at 280 nm for tryptophan fluorescence quenching. Fluorescence spectra were read between 300− 400 nm with 10 nm of emission slit. Appropriate controls containing native (untreated proteins) were run, and corrections were made wherever necessary. All fluorometric spectral studies were carried out at pH 7.4 and room temperature.

Statistical analysis
The data were examined to utilize SPSS version 22 statistics software. The results are illustrated as the mean ± standard error of the mean (SEM). P values <0.05 were supposed statistically significant. The results shown are mean values of three independent experiments done in triplicate under the same conditions to determine the accuracy of the results and to ensure their reproducibility.

OH) by proflavine upon illumination with fluorescent light
The possibility of reactive oxygen species like the hydroxyl radical (⋅OH) being involved in the Fenton reaction was investigated, especially in the presence of metal ions like Cu (II), which is known to engage in the reaction. As shown in Fig. 1A, the formation of hydroxyl radical by light illuminated proflavine with or without Cu (II) was monitored. Increased proflavine and copper concentrations both increased the generation of hydroxyl radicals (Fig. 1B). Various hydroxyl radical scavengers were utilized in the reaction to monitor the specificity of the hydroxyl generation. When deoxyribose degradation was performed in the presence of several hydroxyl radical scavengers such as thiourea, sodium formate, and mannitol, sodium formate showed the greatest inhibition of 46 percent, followed by mannitol, and very little with thiourea (Fig. 1C). When Cu (II) was added to the reaction mixture with proflavine, the inhibition was stronger.

Protein degradation mediated by photo-illuminated proflavine in presence of copper
BSA induced a steady decrease in the synthesis of acid-soluble material after irradiation with proflavine or proflavine plus Cu (II) in fluorescent light, as measured by trypsin digestion assay. However, in the absence of light, no such effect was observed ( Fig. 2A). Furthermore, in the reaction buffer, a decrease in the tryptic hydrolysis of the proflavine-treated protein was seen in a concentration and timedependent manner ( Fig. 2A, B). Furthermore, increasing Cu (II) concentration appears to improve protein modification, as seen by a decrease in protein breakdown evaluated spectrophotometrically by trypsin assay (Fig. 2C).

Proflavine, with or without Cu(II), alters protein structure
BSA was incubated with increasing doses of proflavine and subjected to light, as shown in Fig. 3A. BSA's SDS-PAGE profile revealed considerable protein alteration and degradation. After five hours of exposure to fluorescent light, cleavage and/or modification of a protein by proflavine resulted in smearing and degradation as the sample near the top of the gels was reduced, followed by a broadening of the band at monomer position due to fragmentation to slightly smaller peptides. Furthermore, the presence of Cu(II) accelerated the breakdown of proteins into smaller fragments (Fig. 3B). In addition, the capacity of increasing photo illumination time with proflavine in the presence or absence of Cu(II) to degrade BSA was examined ( Fig. 3C and D). When the light exposure period to proflavine was doubled, there was a significant increase in protein breakdown. Increased light exposure duration to proflavine in the presence of Cu(II) appears to degrade the protein more robustly than in the absence of Cu(II). BSA was not degraded in the absence of light or Cu (II) alone (data not shown).

Photo-illuminated proflavine disrupts enzyme activity
We investigated whether photo-illuminated proflavine with or without Cu (II) disrupted the enzymatic activity of trypsin, a well-known proteolytic enzyme. The release of acid-soluble peptides from the protein is examined to see if the enzyme is inactivated. Before incubation with BSA (as a substrate), trypsin was treated with proflavine and exposed to fluorescent light (Fig. 4A). The activity of trypsin was lowered by about 40 % after 35 min of exposure to light and proflavine. Proflavine in the reaction mixture was likewise found to suppress trypsin activity in a concentration-dependent manner (Fig. 4B). Cu (II) was added to the photo-illuminated process and caused a quick decrease in enzyme activity after 35 min; however, increasing the Cu (II) concentration did not impact the activity. This could be attributed to an increase in the molar ratio of Cu (II) to proflavine (Fig. 4C). When bathocuproine was introduced to the Cu (II)-containing process, the enzyme inactivation was greatly reduced (Fig. 4D), showing that Cu (I) is involved in the photo-illuminated damage to trypsin.

Degradation of trypsin protein and electrophoretic Analysis
In a time-dependent way, photosensitized proflavine appears to cause fragmentation and degradation of trypsin protein. The band intensity of the main 24 kDa subunit of trypsin appears to be decreasing as the incubation period with proflavine increases (Fig. 5A). When the enzyme was treated with proflavine-Cu (II), it degraded faster than when it was incubated without Cu (II). As the incubation period was prolonged, the degradation of the enzyme increased as well, as seen by a decrease in the protein's band intensity. Following that, we looked at the role of free radicals like ( 1 O 2 , 3 O 2 , and • OH) in the degradation of trypsin protein. We did this by incubating the trypsin plus proflavine reaction mixture with free radical scavengers including sodium azide, potassium iodide, and thiourea. Surprisingly, all of these scavengers appear to protect the enzyme from destruction (Fig. 5B). Additionally, the scavenging effects of free radicals were investigated when trypsin was treated with proflavine in the presence of Cu (II). Proflavine and/or Cu (II) increased trypsin degradation, but when those hydroxyl radical scavengers were added, the degradation was reduced, as the band intensities were found to be similar to controls (Fig. 5C).

Fluorescent quenching studies on protein using proflavine
With proflavine concentrations ranging from 0 to 20 μM, the fluorescence quenching of BSA was measured (Fig. 6). As the quantity of proflavine grew, so did the amount of fluorescence quenching. When 20 μM proflavine was incubated with BSA, about 50 % of the fluorescence was quenched. Because we predicted proflavine to attach to BSA near or on tryptophan residues, we examined the binding spectra of proflavine to BSA and tryptophan alone (Fig. 7A, B). We also studied fluorescence quenching in lysozyme and trypsin, two additional proteins with different tryptophan contents (Fig. 7C). When compared to the fluorescence of the free amino acid, all of these proteins demonstrated a change in the maximum wavelength for tryptophan fluorescence to a lower wavelength. The fluorescence quenching for BSA and lysozyme was 49 % and 56 %, respectively, demonstrating that the quenching increased as the protein's tryptophan residues increased. The quenching was shown to increase with increasing proflavine concentration in all cases. The effect of proflavine with or without Cu(II) on the fluorescence spectra of trypsin was also investigated. In the case of unbound amino acids, there was a shift in the maximum fluorescence wavelength to a lower wavelength (Fig. 7D). Free tryptophan quenches fluorescence by 63 percent when combined with proflavine or proflavine plus Cu(II) (Fig. 7A). This demonstrates that BSA is one of the restricted proteins to which proflavine can bind that influences tryptophan residue fluorescence.

Discussion
Proflavine has been linked to several potentially harmful side effects, including mutagenicity, apart from its widespread usage as an antiseptic and disinfectant. Proflavine and its acridine derivatives are shown to infiltrate cells and attach to protein/enzyme and DNA, causing physiological dysfunction and cell death [39,40]. When exposed to visible light, proflavine releases a variety of reactive oxygen species, which cause double-stranded breaks in DNA [6,14].
Previously, we observed that light-induced proflavine generated superoxide ion (O 2 •− ), and that caused structural changes in proteins which were amplified when the divalent metal ion Cu (II) was added [15]. In this paper, we describe hydroxyl radical as a major source generated by light-activated proflavine in presence of Cu (II), which has detrimental effects on enzyme activity and structural alterations in the protein. A robust increase in the synthesis of hydroxyl radical ( • OH) corresponding to an increasing concentration of photo-activated proflavine and Cu(II) was observed, even in the absence of any macromolecules. This is in contrast to the majority of prior investigations, which have indicated that free radical generation from light-induced proflavine is only achievable in the presence of macromolecules like DNA [4]. The specificity of the formation of • OH, radicals in the reaction are validated using hydroxyl radical specific inhibitors.
Copper was employed in our research since it is an essential trace element that is found throughout the body. It's present in a range of tissues, including the liver and kidney, where it's abundant [16]. In the field of biochemistry, metal particles are employed for drug development, diagnostics, delivery, bioimaging, and treatments [41]. Many metalloproteins, including ceruloplasmin, Cu, Zn superoxide dismutase, cytochrome C oxidase, tyrosinase, and ascorbate oxidase, have copper as the redox-active core. Metal ions, such as Cu(II), are known to engage in Fenton-like reactions that stimulate the generation of ROS. During photo-illumination of proflavine, the presence of Cu (II) increased the formation of hydroxyl radicals. Proteins are the primary targets of reactive oxygen species, which can lead to oxidation and influence cellular remodeling and growth [42]. Under aerobic conditions, BSA got fragmented when exposed to • OH and O 2 •− [43]. We investigated the effect of reactive oxygen species produced by photo-illuminating proflavine on BSA. The protein was significantly degraded by proflavine when exposed to light irradiation. The inclusion of Cu(II) in the reaction accelerated the rate of degradation substantially. As our results indicate, photo-illuminated proflavine generated significant structural and Previous studies have shown that in vivo and in vitro interactions of proteins with oxygen free radicals resulted in increased hydrophobicity due to amino acid modifications, potentially increasing proteolysis susceptibility [43][44][45][46]. Furthermore, Wolff and Dean [44] demonstrated that ⋅OH attacked proteins which caused conformational changes in the proteins, increasing their vulnerability to enzymatic proteolysis. However, interestingly, in this investigation, BSA susceptibility to tryptic proteolysis was reduced in the presence of proflavine and Cu(II), indicating the possibility of alteration of certain amino acids, such as lysine and arginine, which are trypsin-recognized amino acid residues. Others have observed that certain amino acids are modified after being exposed to radicals [47], confirming our findings. We previously postulated two putative ROS formation pathways from photoexcited proflavine: triplet ( 3 O 2 ) and singlet ( 1 O 2 ) radicals, and peroxide radicals (O 2 •− ), which may produce (⋅OH) radicals. All of these oxygen and hydroxyl radicals are thought to play a potential role in protein breakdown [15].
Many enzymes accumulate catalytically inactive or less active, more heat-labile forms, which is an indicator of cellular aging [48,49]. Since proflavine inhibits substrate binding by binding competitively to trypsin [50]. Therefore, we used photo-activated proflavine in the presence of Cu (II) to inactivate the monomeric non-glycoprotein enzyme, trypsin. The rate of formation of the enzyme-inhibitor (proflavine) complex is equivalent to that of the enzyme-substrate complex, thus a high concentration of substrate is required for the production of the enzyme-substrate complex when an inhibitor such as proflavine is present. For this reason, BSA, the trypsin substrate in our investigation, was always utilized in excess. Despite this, trypsin's proteolytic activity was shown to be significantly reduced, most likely due to free radical-mediated damage to specific amino acids in the protein caused by proflavine. When Cu (II) was added to the process, the loss of trypsin activity was even more significant.
The steady decrease in emission intensity demonstrated that proflavine either directly binds to tryptophan or alters the conformation of BSA, resulting in tryptophan shielding, as revealed by the emission spectra of BSA recorded after the addition of increasing proflavine concentrations. Changes in the aromatic side-chain composition, such as a loss of conjugation from tryptophan or other aromatic amino acids, are reflected in the decreased fluorescence [51]. For fluorescence investigations, BSA and other proteins with different levels of tryptophan, such as lysozyme and trypsin, were employed. In all cases, a change in λ max for tryptophan fluorescence was detected, along with quenching, indicating that proflavine binds to these proteins around tryptophan residues. The shift in λ max was maximum with BSA followed by lysozyme and minimum with trypsin. This indicates that trypsin's tryptophan is in the least hydrophobic environment possible.
Our study is significant since a wide range of proflavine and acriflavine compounds have been produced and used clinically as antibacterial, antimalarial, and antiprotozoal medicines, [4,11,52]. In addition, work on developing novel proflavine compounds and derivatives have begun, which could have interesting anticancer properties [53].
Taken together, photoinduced-proflavine seems to generate an excess of hydroxyl radicals that resulted in protein degradation and loss of enzyme activity. Photoexcitation of proflavine in the presence of metals ions especially Cu(II) augmented the generation of hydroxyl radicals. Proflavine resulted in oxidative modifications of protein and enzyme structure and function. The findings of this study might contribute to a better understanding of proflavine's harmful effects on protein and enzyme breakdown caused by oxygen-free radicals. However, further research into proflavine's harmful role in oxidizing and destroying proteins and enzymes is needed, as this substance may increase the chance of acquiring a variety of disorders.