Skin detoxifying peptide with enzymatic activity

Download PDF version of article

Most cosmetic problems have their roots in skin cutaneous condition. The skin and more precisely, the stratum corneum that covers externally the whole cutaneous surface is our shelter against the external world.

So it is not difficult to ascertain that the skin will be in charge to slow down the external aggressions that may come from the environment. The environmental conditions that surround us have changed with the time, otherwise the skin structure and its functions remain the same. The skin keeps its mission of protecting and defending not only against the cold or the heat but against pollution, a damaged atmosphere and against a stressful lifestyle, conditions which are far from the natural models. However, the skin has to fight to keep itself in optimal condition. Apart from toxins that are externally generated, there exists another issue coming from the endogen toxins, as protein denaturalisation. So it is not surprising that at certain moments the skin suffers from this daily fight. Infinitec Activos S.L. launches a new and revolutionary cosmetic treatment, based on a combination of two actives, one from natural origin and the other one a synthetic peptide, that deactivates the free radicals generated by the environment as well as the free radicals generated in the skin (avoiding proteosomic degradation). By this method the cellular regeneration is enhanced as well as the anti-ageing effect.

The air of our cities accumulates toxic substances such as carbon monoxide and sulphur dioxide from the exhaust pipes of vehicles and from the chimneys of the houses and factories, that irritate the dermis of our skin. Another factor that causes skin ageing and is also related to the air that surrounds us is the effect of free radicals. The air is principally composed of a mixture of gases such as nitrogen, oxygen and water vapour. The oxygen, which accounts for 21% of the composition, can be particularly harmful when it is at its most dangerous as hydroxyl and superoxide. Exposure to ultraviolet rays also generates a good amount of these free radicals, as well as exposure to various types of radiation, including X-rays. Also, some foods, such as red meat, smoked fish, coffee, fats, and especially fried and overheated oil can be a source of free radicals. To fight against this effect, nature has given Man, and by extension all living organisms, some mechanisms that are able to scavenge free radicals and purify the skin and the body in general. These mechanisms are due to the enzymatic activity of peroxidase, superoxide dismutase and catalase. Furthermore, the protective action offered by various antioxidants such as vitamin C, vitamin E, -carotene, flavonoid, selenium, zinc, lycopene, copper, etc. If these mechanisms are altered, organic alterations occur as a consequence, such as the destruction of proteins (which bring support to the skin tissue), lipids (fats that provide the nutrients to skin cells) cell mitochondria (responsible for cellular respiration), and even at DNA level. It is easy to understand that if the mechanisms and protection from free radicals are severely affected, they damage the structure of skin cells and skin ages rapidly.

Prevention and purifying effect of peroxidase

The peroxidase enzyme plays an important protective role against the damage caused by oxidation in the skin. Infinitec S.L. has designed a peroxidase mimetic synthetic peptide in order to develop a protective agent and skin purifier (against oxidative damage caused by environmental toxicity). In an abnormal physiological state or during the ageing process, the cells are unable to completely get rid of the excess free radicals. Hence the need for the action of peroxidases trying to balance and eliminate the metabolism of free radicals. Despite the importance of peroxidases in the prevention of ageing, skin detoxification and therapy of diseases caused by free radicals and environmental pollution, this protein is found in very limited amounts naturally in the skin. Thus, a gradual dosing of this enzyme becomes necessary. However, this protein is difficult to produce in either a medium or large scale. In accordance with the literature so far no known molecule is able to mimic the structure of the enzyme peroxidase. The deficiency of this enzyme in the skin and generally on the body can cause various damage and premature ageing. These can be:

•  DNA damage level, causing alteration of the nucleotide bases, and sister chromatid exchange and defective linking in the DNA and protein. This leads to mutations and loss of cellular control.
•  Another associated disorder would be lipid peroxidation, a chain reaction in which an OH group removed a hydrogen atom from a methylene group of a fatty acid of the cell membrane, transforming it into a new free radical (peroxy radical). This, in turn, removes a methylene hydrogen atom from a neighbour fatty acid. This process is repeated until chain cell death.
•  Damage to proteins by altering the conformation and function of proteins due to the oxidation of specific amino acids as lysine, serine, arginine and proline. The loss of function results in a metabolic process slowdown and it induces the production of antibodies.
•  Redox signal – altered redox balance changes intracellular signals inducing proliferation, apoptosis, genetic alterations and necrosis.

 Results and discussion

Synthesis and characterisation of mimetic peptide

The mimetic peptide was prepared by solid phase methodology using the strategy Fmoc/t-Bu. Activators for synthesising various combinations were used such as DIC/HOBt, TBTU/HOAt or HATU/HOAt. The peptide was cleaved from the resin and purified by RP-HPLC. Finally, the product was characterised by mass spectrometry and amino acid analysis. The mimetic peptide was characterised by RP-HPLC on a Waters 996 photodiode array detector instrument. This instrument is equipped with a Waters 2695 modular separator and Millenium chromatography software. The column was C18 reverse phase (reverse-phase Symmetry C18 HPLC columns, 4.6 x 150 mm, 5 m) (Waters, Ireland). The peptide was detected in UV at 220 nm and a linear gradient of 5 to 100% CH3CN (0036% TFA) and H2O (0045% TFA) in 8 min at a flow rate of 1.0 mL/min. The peptide was analysed by MALDI-TOF and ES (+)–MS on an Applied Biosystems Voyager DE RP, using a matrix 2, 5-dihydroxybenzoic acid (DHB), and a spectrometer of Micromass VG-quattro.

Breakdown of H2O2

The activity of the mimetic peptide was measured by H2O2 degradation using a specific assay of ascorbate peroxidase. Its specific activity is 4.0 x 101 U/moL with H2O2 as substrate (1 L). The mimetic peptide was capable of catalysing 1 g of ascorbate per minute (see Table 1). With 0.5 mM of ascorbate in 50 mM of PB at pH 7, the kinetic of reaction on H2O2 exhibited an hyperbolic behaviour with KM at 0.98 M, following Michaelis-Menten kinetics. The value of Kcat/KM was 1.64E-04. This study shows the great enzymatic capacity of Detoxiquin for exogenous detoxification of H2O2.

Detoxifying assay over time (Xylenol assay)

H2O2 concentrations were measured using the xylenol orange assay (A560) (Gay & Ebicki 2000).Working reagent contained 0.1 mL of reagent A comprising 25 mm FeSO4, 25 mM (NH4)2SO4 and 2.5 m H2SO4, and 10 mL of reagent B comprising 125 mM xylenol orange (Sigma) and 100 mM sorbitol. Assays contained 0.1 mL of the sample solution (Detoxiquin mimetic peptide, 10 nm; vitamin E, 100 M; and negative peptide 100 M) and 3 mL of the working reagent. The rates of exogenous breakdown of 500 m of H2O2 were measured. The concentration of H2O2 that remained in the solution was measured after 60 min. The pH of all solutions was adjusted to 7.0.

Results

The rates of exogenous detoxification of H2O2 by Detoxiquin mimetic peptide was significantly higher than vitamin E over time. Besides, while one molecule of mimetic peptide can be recycled and break down various molecules of H2O2, vitamin E can only break down one molecule of H2O2.

Prussian blue staining

Detoxiquin Mimetic Peptide (now referred to as ‘the peroxidase mimetic peptide’) not only acts in an extracellular manner but also eliminates free radicals generated intracellularly. Prussian blue staining, a technique which allows iron (Fe) detection was used to prove the presence of Fe in the peroxidase mimetic peptide. In order to carry out Prussian blue staining, cells were incubated with our compound for 4 hours and cells without the peroxidase mimetic peptide. After 4 hours, cells were air dried for 24 hours. Cells were subsequently fixed with (cold) acetone for 10 minutes and later on they were incubated with fresh solution of potassium hexacyanoferrate [II] (Sigma, Taufkirchen, Germany; 2%, with 0.1 N HCl) for 10 minutes. Cells were revealed afterwards with nuclei red marker (Merck, Germany). As shown in Figure 2, the peroxidase mimetic peptide acts at extra and intracellular levels, guaranteeing the elimination of free radicals and harmful ions in cells.

Determination of Fe complexation in the peroxidase mimetic peptide

The enzymatic activity of the peroxidase mimetic peptide depends to a great extent on the presence of Fe in the molecule. Also, the mimetic peptide serves as a carrier of iron to the skin and the body in general. Furthermore, the presence of Fe provides magnetic properties to the final product, which improves the quality of the skin by purifying the harmful positive ions in the skin. The existence of Fe in the peptide and of its magnetic properties was determined by means of a MRI (magnetic resonance imaging). As it is shown in Figure 4, the presence of Fe in the the peroxidase mimetic peptide sample generates a contrast image which is created by different relaxation times (T1, T2). On the other hand, the lower the intensity, the higher the content of Fe is present in the molecule. These results mean that the peroxidase mimetic peptide presents magnetic properties.

Conclusion

Due to the nature of the enzyme protein, combinatory chemistry becomes a very useful tool to mimic endogenous molecules. At Infinitec, a peroxidase mimetic peptide has been developed for its ability to neutralise the harmful effects of free radicals. In addition to its peroxidase activity, the peptide has the unique ability to regenerate its own activity, which does not happen in other antioxidants which are consumed during the reaction, and it can enhance its protective effect on the skin.

References

1 Riebe O, Fischer RJ, Wampler DA, Kurtz DM Jr, Bahl H. Pathway for H2O2 and O2 detoxification in Clostridium acetobutylicum. Microbiology 2009; 155 (Pt 1): 16-24. 2 Minibayeva F, Kolesnikov O, Chasov A et al. Wound-induced apoplastic peroxidase activities: PPCC their roles in the production and detoxification of reactive oxygen species. Plant Cell Environ 2009; 32 (5): 497-508 3 Kalyani DC, Telke AA, Dhanve RS, Jadhav JP. Ecofriendly biodegradation and detoxification of Reactive Red 2 textile dye by newly isolated Pseudomonas sp. SUK1. J Hazard Mater 2009; 163 (2-3): 735-42. 4 Schäfer M, Werner S. Oxidative stress in normal and impaired wound repair. Pharmacol Res 2008; 58 (2): 165-71. 5 Anastasi A, Coppola T, Prigione V, Varese GC. Pyrene degradation and detoxification in soil by a consortium of basidiomycetes Isolated from compost: Role of laccases and peroxidases. J Hazard Mater 2009; 165 (1-3): 1229-33 6 Hamelet J, Seltzer V, Petit E et al. Cystathionine beta synthase deficiency induces catalasemediated hydrogen peroxide detoxification in mice liver. Biochim Biophys Acta 2008; 1782 (7-8): 482-8 7 Corstjens H, Declercq L, Hellemans L, Sente I, Maes D. Prevention of oxidative damage that contributes to the loss of bioenergetic capacity in ageing skin. Exp Gerontol 2007; 42 (9): 924-9 8 Afaq F, Mukhtar H. Effects of solar radiation on cutaneous detoxification pathways. J Photochem Photobiol B 2001; 63 (1-3): 61-9. 9 Steiling H, Munz B, Werner S, Brauchle M. Different types of ROS-scavenging enzymes are expressed during cutaneous wound repair. Exp Cell Res 1999; 247 (2): 484-94.