Biochemical Characterization of Protease and Its Impact By Nano Particles in Sera of Iraqi Patients with Burns

Sura Ahmed Abdulsattar,Dhia Hadi Hussain,Hasan Hadi Algam
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Keywords : Burn, protease, gold nanoparticle, nickel nanoparticle, immunoglobulin
Medical Journal of Babylon  12:4 , 2016 doi:1812-156X-12-4
Published :22 January 2016

Abstract

Proteases have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. The present study aims to characterization of protease and evaluate their impact by Gold Nano Particles and Nickel Nano Particles. A total of 30 patients with burn and 25 healthy individual with matches were included in this study. Gold nanoparticle and nickel nanoparticle were prepared using Pulsed Laser Ablation in Liquid method .The activity of serum protease was determined using casein as substrate. The results indicated increased protease activity in sera of burn patients and inhibition effects of both gold and nickel nanoparticles on protease activity. Thermodynamically favorability of the reaction can depend on the temperature. We conclude that nanoparticles such as gold and nickel can be used as treatment of burns through their role in homeostasis due to their inhibition impact on protease activity.

Introduction

Aburn is a type of damage to skin caused by heat, chemicals, electricity , radiation or friction, [1]. Burns are one of the most destructive of all injuries and a major global crowd health turn [2-4].They are the fourth most popular type of hit worldwide, following traffic accidents, falls, and interpersonal violence [5,6]. Burns result in about two million physician visits per year [7]. Approximately 90 percent of burns occur in low to middle income countries, regions that generally lack the substantial infra-structure [8,9].Burn injuries encompass the partial or complete devastation of the integumentary system: the skin. The layers of the skin are ruined and these results in local and systemic disorders. When the skin is damaged by a burn, it may result in compromised immunity, hypothermia, increased fluid loss, infection, changes in appearance, function, and body image [10].Inflammation is a typical and early response of a burns tissue. It results in an increase in the number of immune cells in the area of damage or infection which remove damaged or dead cells and initiate the healing process which involve conversion of an inactive proteolytic enzyme into an active enzyme[11].These enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are widely applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unfavorable proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics [12]. Gold nanoparticles (GNPs) with controlled geometrical, optical, and surface chemical properties are the subject of intense studies and applications in biology and medicine. To date, the ever increasing variety of published examples has included genomics and biosensorics, immunoassays , clinical chemistry, targeted delivery of drugs, and antigens, photo thermolysis of cancer cells and tumors and optical bioimaging of cells and tissues with state-of-the-art nanophotonic detection systems [13].Nickel nanoparticles (NNPs) has become one of the interesting materials in research communities due to the varied promising applications in the field of catalysis [14-16] , and magnetism [17].Biomedical applications of Nickel nanoparticles can be categorized according to their application inside(in vivo) or outside the body (in vitro). The main use in vitro applications, is in diagnostic, separation, and selection, while in vivo applications, it could be further separated in therapeutic and diagnostic applications (nuclear magnetic resonance [NMR] imaging) [18-20]. The present study aims to characterize of protease kinetically and thermos-dynamically and evaluate the GNPs and NNPs effect on protease activity as example of inflammatory proteins in sera of patients with burns.

Materials and methods

A total of 30 patients with burn attending Al-Kindy Hospital in Baghdad city and Medical City Hospital burns specialist were participated in this study. We obtained general information about each patient, including age, sex, etiology, location of burns, degree burn. As a control of 25 healthy individual with matches were included in this study. Five ml were collected from healthy donors and patients. The blood sample was centrifuged at 3000 rpm for 5 min after allowing the blood to clot at room temperature. Serum separated and transferred into test tube, and stored at -20oC until being used. Gold nanoparticle and nickel nanoparticle were prepared using Pulsed Laser Ablation in Liquid method[21].Structure and nanosize measurement of nanoparticles samples were identified by the Scanning Electron Microscope (SEM), Atomic Force Microscope (AFM). Absorbance spectra of NPs solution was measured by UV-VIS double beam spectrophotometers The activity of serum protease was determined using casein as substrate according to assay method of Isshaya, et al [22],with modification [23].
 Thermodynamic parameters of protease were determined using Arrhenius plots (1/T ×1000 vs. log of activity) of casein. Energy of activation (Ea)was calculated from the curve slope, other thermodynamic parameters, such as, free energy change (?G), entropy change (?S), enthalpy change (?H) were calculated using the following equations: [24]

Log Vmax = log A- Ea /2.303 R × 1/T
slope=Ea/2.303R

Enthalpy change ?H was calculated from the following equation:
?H*= Ea –RT
The Free Gibbs energy ?G was calculated from the following equation:
?G* = -RT Lin Vmax + RT Lin (KT/ h)
Change of entropy was calculated from the following equation:
?S*=(?H*-?G*)/T
The statistical software (SPSS v 19; Chicago, IL, USA) was used. The data were analyzed using unpaired t-test and person correlation coefficients. Differences were considered significant when P< 0.05.




Results

An interesting aspect of metal NPs is that their optical properties depend strongly on the particle size and shape. As an example, bulk gold looks yellowish in reflected light, while thin gold films look blue in transmission. This blue color steadily changes to orange as the particle size is decreased to ~ 3 nm. These effects are the result of changes in the Surface Plasmon Resonance (SPR) [25]. Figures(1a,and 1b) show the Surface Plasmon Resonance absorption spectrum of colloidal solutions of GNPs, and NNPs respectively. The pulse energy at the target surface was 700 mJ/Pulse for GNPs, which shows the absorbance peaks with broad band around 530nm, 465nm, and 195nm(Figure 1:a). We observed a faint pink coloration of the solution after several pulses of the experiment, as shown in optical picture. In the absorption spectrum of the solution, the surface plasm on related peak could be clearly distinguished. The peaks were around 195?530 nm These peaks were consistent with the presence of small 20?100 nm particles in the colloid, which confirmed by AFM and SEM, as compared to other research, which showed the presence of small 3?30 nm particles in the colloid [26].(Figure 1:b) which shows the absorbance peaks that occur at around 195 nm is the characteristic of NNPs[27]. The pulses energy at the target surface was 700 mJ/Pulse, The NNPs was transparent tends to faint black in color as shown in optical picture.

Discussions

An interesting aspect of metal NPs is that their optical properties depend strongly on the particle size and shape. As an example, bulk gold looks yellowish in reflected light, while thin gold films look blue in transmission. This blue color steadily changes to orange as the particle size is decreased to ~ 3 nm. These effects are the result of changes in the Surface Plasmon Resonance (SPR) [25]. Figures(1a,and 1b) show the Surface Plasmon Resonance absorption spectrum of colloidal solutions of GNPs, and NNPs respectively. The pulse energy at the target surface was 700 mJ/Pulse for GNPs, which shows the absorbance peaks with broad band around 530nm, 465nm, and 195nm(Figure 1:a). We observed a faint pink coloration of the solution after several pulses of the experiment, as shown in optical picture. In the absorption spectrum of the solution, the surface plasm on related peak could be clearly distinguished. The peaks were around 195?530 nm These peaks were consistent with the presence of small 20?100 nm particles in the colloid, which confirmed by AFM and SEM, as compared to other research, which showed the presence of small 3?30 nm particles in the colloid [26].(Figure 1:b) which shows the absorbance peaks that occur at around 195 nm is the characteristic of NNPs[27]. The pulses energy at the target surface was 700 mJ/Pulse, The NNPs was transparent tends to faint black in color as shown in optical picture.

Conclusions

We conclude that nanoparticles such as gold and nickel can be used as treatment of burns through their role in body homeostasis due to their inhibition impact on protease activity .

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