the effects of gold and silver nanoparticles on ...the effects of gold and silver nanoparticles on...

THE EFFECTS OF GOLD AND SILVER NANOPARTICLES ON TRANSAMINASE ENZYMES ACTIVITIES

1Salma Abdul Rudha Abbas,1Amer Hasan Abdullah,1Shaemaa Hadi Abdul Sada,2Abdulrahman K.Ali

1Department of Chemistry;College of Science;Al-Mustansiryh University 2School of Applied Sciences – University of Technology

Abstract Gold and silver nanoparticles colloids were produced by irradiating a metallic target plates with a thickness of 1mm immersed in distilled water with a pulsed laser beam. The size and size distributions of the metals nanoparticles were examined by the transmission electron microscope TEM analysis. The nanoparticles concentrations were also characterized by atomic absorption spectroscopy AAS measurement. The effects of gold and silver nanoparticles were studied on the activities of GOT and GPT enzymes in the sera. Gold and silver nanoparticles demonstrated inhibitor effects on the (GOT) and (GPT) activities, and these effects increased with increasing the concentrations of the nanoparticles. Keywords: GOT,GPT, Inhibition, Nanocolloids Introduction Laser ablation of bulk target immersed in liquid environment which is simple method, recently has attracted much attention for nanoparticles formation [1-4]. Nanomaterial display unique, superior and indispensable properties and have attracted for their distinct characteristics are unavailable in conventional macroscopic materials. Their uniqueness arises specifically from higher surface to volume ratio and increased percentage of atoms at the grain boundaries. They represent an important class of materials in the development of novel devices that can be used in various physical, biological, biomedical and pharmaceutical applications [5-8]. In view of the importance of aminotransferase enzymes reactions like Glutamate Oxaloacetate Transaminase (GOT), Glutamate Pyruvate Transaminase (GPT) which form links between the metabolism of amino acids, carbohydrates and fats

Glutamate Oxaloacetate Transaminase (GOT) activity (EC 2.6.1.1) :- It's called also aspartate aminotransferase (AST), is one of the most important of transaminases enzyme, and catalyzes the transfer of the amino group of aspartate to α-ketoglutrate. GOT is widely distributed in human tissues; heart, liver, skeletal muscle and kidney. The optimum conditions of maximum enzyme activity are pH=7.4 , temp. = 37cº. The enzyme stability 3 days in 25 cº , 1 week in 5 cº and 1 month in -25 cº. The clinical usefulness of the enzyme is largely restricted to the diagnosis of heart and liver diseases. Large amount of GOT may be released into the blood. Very high levels are observed in acute liver disease while lesser elevation is seen in chronic liver disease [9-11]. Glutamate Pyruvate Transaminase (GPT) activity( EC 2.6.1.2) :- It's also called alanine amino transferase (ALT) which is prevalent in mammalian tissue catalyzes the transfer of the amino

Amer Hasan Abdullah, et.al., Int. J. Chem. Res., 2011v01(4), 1-11 ISSN: 2249-0329

IJCSR |SEP - OCT 2011 Available [email protected]

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group of alanine to α-ketoglutrate.GPT found in a highest concentration in liver in spite of its active occurrence in skeletal muscles, heart and kidney. The GPT activity in tissues is generally less than GOT. Significant elevation of S.GOT occurs in sever acute hepatitis where the enzyme is released in to the circulation. GPT level is found to increased in the following diseases; infection hepatitis, liver cirrhosis and biliary cirrhosis, obstructive jaundice, liver cancer [11-14]. Materials and methods 1-Nanoparticles Preparation Gold and silver nanoparticles colloids were produced by irradiating a metallic target plates with a thickness of 1mm immersed in distilled water with a pulsed laser beam. The ablation was performed with the (1064 nm) of a Nd:YAG laser (HUAFEI) operating at 10 Hz repetition rate, with a pulse width of 10 ns. The beam was focused on the surface of the target through a lens with 11cm of focal length. The spot size was about 1.5mm in diameter. The size and size distributions of the metals nanoparticles were examined by the transmission electron microscope TEM analysis, using a CM10 pw6020, Philips-Germany. UV–vis absorption spectroscopy measurements were carried out on a double beam, CECIL C. 7200 (France) spectrophotometer. The nanoparticle concentrations were also characterized by Atomic absorption spectroscopy AAS measurement (model GBS 933, Australia), was carried out for the prepared samples.

2-Effects of gold and silver nanoparticles colloids on GOT and GPT activities:- Colorimetric determination of GOT or GPT activity according to the following reactions:- L-Aspartate +α-ketoglutrate GOT Oxaloacetic+glutmate Alanine + α-ketoglutrate GPT pyruvate+glutmate The pyruvate or oxaloacetat formed was measured in its derivated form 2,4-dinitrophenylhydraone, which was absorbed at wave length 505 nm [15-17] . A-A stock colloid (15 µg/mL) concentration of gold nanoparticles and (19 µg/mL) concentration of silver nanoparticles and then the following concentrations (1,3,6,9,12) µg/mL of gold nanoparticles and (1,3,6,9,12,15) µg/mL of silver nanoparticles are prepared. The different concentrations of the nanoparticles are prepared by diluting with deionized water using the stock colloid. B-The enzymes GOT and GPT activities were measured in human serum by using the same methods of these enzymes with replace 100µL of deionized water with 100µL of gold or silver nanoparticles colloids. The inhibition percentage was calculated by comparing the activity with and without the gold or silver nanoparticles colloids and under the same conditions, according to the equation:- %Inhibition= 100-100 x Activity in the presence of inhibitor Activity in the presence of inhibitor C-A constant concentration of Au and Ag nanoparticles (12 µg/mL) was used with different substrates



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concentrations of GOT and GPT (40,80,120,160,200)mmol/L. Buffers were used to prepare different substrates concentrations of these enzymes (phosphate buffer pH 7.4,100mmol/L) .The enzymes activities were determined with and without the nanoparticles, by using the Lineweaver-Burk equation and plotting 1/v against 1/[s] were evaluated values[ki ,Apparent vmax (vmapp) , Apperent km (kmapp) ,Type of inhibition] [18]

Results and discussion This research addresses on preparation of pure noble metals of Au and Ag nanoparticles colloids, which have significant biological and chemical effects [5-8,19-22], and investigation of the effects on activities of (GOT and GPT) enzymes. Fig. 1(A and B), shows the extinction spectra of colloidal solutions of Ag and Au samples, respectively. The Nd-YAG laser of 1064 nm was utilized as an ablation source. The pulse energy at the target surface was varied in the range (300-600 mJ) and the beam was focused to have a diameter near the outer edges of the target of 1.27 and 0.85 mm for Ag and Au, respectively. The metal plate was fixed in a glass vessel filled with 1 ml DDDW thus the smokelike colloids above the metal plate was observed. The plate was located at 8 and 7 mm from the liquid surface for Ag and Au, respectively. Laser ablation listed for 15 pulses and the solution gradually turned to colored with the increase of the number of laser pulses. Fig. 1(A) shows the Absorbance peaks that occurred at around 400 nm is the characteristic SPE signature of Ag

nanoparticles[23]. Fig 1-B shows broad band with the Absorbance peak around 526 nm with the peak position remaining practically constant, that indicates the production of gold nanoparticles[24]. We observed a faint pink coloration of the solution after several pulses of the experiment. In the absorption spectra of the solutions, the surface plasmon related peak could be clearly distinguished. This peak was around 520–530 nm, which was consistent with the presence of small 3–30 nm particles in the colloid [25], which also confirmed by TEM. Figure 2(A and B) shows the TEM images and corresponding size distributions of silver and gold nanoparticles, produced by laser ablation of silver plate immersed in pure water. The nanoparticles thus produced were calculated to have the average diameters of 14 nm. It is observed that the average diameter and size distribution was increased with the increase of the laser energy. The origin of the surface morphology of the irregularly shaped particles sizes and the size distribution broadens can be explained by absorption by defects and thermally induced pressure pulses which cause cracking[26]. The biochemical tests revealed that nanoparticles of Au and Ag caused inhibitory effects on GOT and GPT enzymes activities, the relationships between nanoparticles of Au and Ag concentrations versus and the activity of enzymes as shown in Figures (3) and (4). These results observed that any increase in nanoparticles of Au and Ag concentrations caused increasing in percentage of



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inhibition of enzymes. The greater inhibition of Au nanoparticles on GOT enzyme was 40.5% at concentration (15 µg/mL) and Ag nanoparticles was 30% at concentration (15 µg/mL), as shown in figures(4),and greater inhibition of Au nanoparticles on GPT enzyme was 69% at concentration (15 µg/mL) and Ag nanoparticles was 14.3% at concentration (12,15,19)µg/mL,as shown in figures (5). Competitive, noncompetitive and uncompetitive inhibition can be easily distinguished with the use of double reciprocal plot of the Lineweaver-Burk plot. Two sets of rate determination in which enzyme concentration was held constant, were carried out. In the first experiment the velocity of enzyme without inhibitor was established, in the second experimental constant amount of inhibitor is included in each enzyme assay. Varieties of substances have the ability to reduce or eliminate the catalytic activity of specific enzyme [18]. Table(1) and figure (7) showed that the kinetic parameters ( Kmapp, Vmapp, and Ki , type of enzyme inhibition) using Lineweaver-Burk plot for Au and Ag nanoparticles on serum GOT activity. The Vmax and Km without Au or Ag nanoparticles were 33.33 U/L, 0.125 mM respectively. A liquate 12 µg/mL of Au and Ag nanoparticles were competitive inhibition for enzyme activity. Competitive inhibition changed the Km but not the Vmax of the enzyme. When concentration of Au or Ag nanoparticles 12µg/mL the Kmapp were (0.166,0.166) mM. By using Lineweaver-Burk equation was calculated the Ki values of enzyme

for Au and Ag nanoparticles which were studied in different concentration. The Ki of Au and Ag nanoparticles were (39,39)mM. Table(1) and figure (8) showed that the kinetic parameters ( Kmapp, Vmapp, and Ki , type of enzyme inhibition) using Lineweaver-Burk plot for Au and Ag nanoparticles on serum GPT activity. The Vmax and Km without Au or Ag nanoparticles were 33.33 U/L, 0.143 mM respectively. A liquate 12 µg/mL of Au and Ag nanoparticles were competitive inhibition for enzyme activity. When concentration of Au or Ag nanoparticles 12µg/mL the Kmapp were (0.25,0.2)mM. By using Lineweaver-Burk equation was calculated the Ki values of enzyme for Au and Ag nanoparticles which were studied in different concentration. The Ki of Au and Ag nanoparticles were (16,30)mM. Heavy metales are toxic and react with proteins,therefore they bind protein molecules, heavy metales strongly interacts with thiol groups of vital enzymes and inactivates them [19].In addition,it is believed that Ag and Au bind to functional groups of proteins, resulting in protein deactivation and denaturation [21,22]. We hypothesized that nanoparticles of Au and Ag interact with functional groups of GOT and GPT enzymes, resulting in protein denaturation and inactivate it, so nanoparticles of Au and Ag inhibited the enzymes . The present work is the first study that demonstrates the effects of gold and silver nanoparticles colloids on the activities of GOT and GPT enzymes. Some recent researches demonstrated that gold nanoparticles colloids can be used



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in diagnosis and treatment of some kinds of cancer [5-8,20,27].Other researches proved that silver nanoparticles colloids are anti-bacterial[19-22]; Therefore, it was useful to know what is the effects of gold and silver nanoparticles colloids on activities of the different enzymes when enter to the human body ,then it would be known what the side effects of gold and silver nanoparticles colloids on the human body is. GOT and GPT are important enzymes which are found in the human body because they are responsible for the metabolism of amino acids. In view of the importance of transaminase

enzymes reactions like GOT and GPT which form links between the metabolism of amino acids, carbohydrates and fats. Activation or inhibition of GOT and GPT by chemicals effects on the metabolism of amino acids, carbohydrates and fats, this research proved that gold and silver nanoparticles colloids inhibition of GPT and GOT enzymes; Therefore, catabolism of amino acids will decrease, then the concentration of amino acids in blood would increase and cause buildup of protein in blood and effect on urea cycle and tricarboxylic acid cycle.

Fig. 1: Absorbance spectra of silver nanoparticles (A), and gold nanoparticles (B), obtained by laser ablation of metal plates immersed in DDDW with laser energy of 600 mJ, laser shots of 15 pulses and wave length is 1064 nm of Nd-YAG.



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Fig. 2: TEM images and size distributions of silver (A), and gold nanoparticles (B), produced by laser ablation of metal plats immersed in pure water, (λ=1064 nm and laser shots of 15 pulses).

Au concentration( µg/mL)

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Figure (3):- GOT enzyme activity as a function of concentration of

(A) Au nanoparticales (B) Ag nanoparticles.

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(B) Ag Nanoparticles



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Figure(6):-% Inhibition of GPT enzyme and (A)Au nanoparticles concentrations (B)Ag nanoparticles concentrations

Lineweaver-Burk plot of GOT activity

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Figure (7):-Lineweaver-Burk plots for (A) Au and

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Lineweaver-Burk plot of GPT activity

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Figure (8):-Lineweaver-Burk plots for (A) Au and (B) Ag nanoparticales effects on

GPT

Table(1):-The kinetic properties of ChE and MAO with Au and Ag nanoparticles.

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