EFFECTS OF COPPER IONS ON RADIOSTABILITY OF BIOPOLYMER
SYSTEMS
1 2 1 2
S. Bayülken , M. Mustafaev , G. Yüce , Z. Mustafaeva
Çekmece Nuclear Research and Training Center, P.O. Box 1, 34149 Atatürk Airport, Istanbul-TURKEY 2 Yıldız Technical University, Department of Bioengineering, 34210 Esenler-Istanbul, TURKEY
ABSTRACT
Water-soluble bioconjugates of synthetic polyelectrolytes (PE) with biomolecules (proteins, polysaccharides, etc.) have important applications in various areas1. Such systems include complexes stabilized by multipoint electrostatic and hydrophobic interactions between the fragments of polyelectrolytes and biomolecules and conjugates in which the functional groups of the components are linked by covalent bonds. A relatively new technique involves the use of transition metal (Cu(II)) compounds as a means of activating the polymer carrier and allowing direct coupling of proteins without prior derivatization of the activated polymer, through formation of chelates.
Developing biopolymer systems having long-term protective effects against radiation and decreasing the radioactive contamination due to a radiation accident or nuclear war, is very important for the health of human being.
In this study, the synthesis of biopolymer systems (radioprotector) and the investigation of their radiostability, toxic and radioprotective effects on model systems are aimed to perform.
Aqueous solutions of ternary PE+Cu2++Bovine serum albumin (BSA) complexes have been irradiated with 60Co y-rays and the resulting changes occurred have been measured by high-performance liquid chromatography (HPLC)2. Addition of Cu2+ ions to polymer-protein mixture has been shown to protect the polymer and protein components against radiation damage.
The effect of antioxidant polymers on radiation will be investigated by the cromosome aberation and micronucleus methods in blood samples.
1. INTRODUCTION
Copper is found in a number of enzymes that play important roles in, for example, electron tansport and antioxidant defense. Copper is an essential trace element involved in a variety of critical metabolic processes. Although copper is the third most abundant essential trace mineral in the body, after iron and zinc, most people consider it unimportant. Even worse, many people have actually taken steps to exclude it from their diets and dietary supplements, believing it to be nothing more than a cause of free radical reactions. In the past seventy years, much has been learned about the important biological roles of copper and the copper- dependent enzymes. In fact, copper is emerging as one of the most important minerals in our diet. Unbound, free copper is not found in large quantities in the human body. Instead, almost all of the copper in our bodies is bound to either transport proteins (ceruloplasmin and copper-albumin), storage proteins (metallothioneins), or copper containing enzymes3. A substantial number of copper metalloenzymes have been found in the human body. Copper is essential for the proper functioning of these copper-dependent enzymes, including cytochrome C oxidase (energy production), superoxide dismutase (antioxidant protection), tyrosinase (pigmentation), dopamine hydroxylase (catecholamine production), lysyl oxidase (collagen and elastin formation), clotting factor V (blood clotting), and ceruloplasmin (antioxidant protection, iron metabolism, and copper transport). Most features of severe copper deficiency can be explained by a failure of one or more of these copper-dependent enzymes.
The total amount of copper in the body of a man weighing 70 kg. is estimated to be about 80 mg.4. The liver is the major location of stored copper, containing about 10 percent of the total-body content5. Maintaining a steady level of copper in the body depends upon a balance between intestinal absorption and biliary excretion. Biliary excretion of copper is capable of substantially increasing when excess copper is ingested. Beneficial effects of a copper-containing organic [Cu(II)2(3,5-DIPS)4] are, for example, reported to increase survival in gamma-irradiated mice6. In this study, P A A p oly(N IP A A m ), B SA , SO D and th eir m ixtures
2. E X P E R I M E N T A L M aterials and M ethod
PAA was synthesized and fractionated as explained in the literature7. PAA was prepared by radical polymerization of aciylic acid in toluene with benzoil peroxide as an initiator. PAA was fractionated from 3 to 4% solution in methanol using fractional precipitation by ethylacetate. The molecular weight of the fraction used in this study was 240 kDa. BSA and neocuproine were purchased from the Sigma, SOD and CuS0 4.5H20 were purchased from Merck. The molecular weights of BSA and SOD are 70 kDa and 31.4 kDa respectively. Poly(NIPAAm) was synthesized as explained in the literature8.
A ll th e solutions w ere p repared 0.1% in ph osp h ate b u ffer at room tem p eratu re. A fter d issolu tion , pH w as ad justed to 7 by 0.1 N N aO H if needed. Solution o f C u S 0 4.5H 20 w as p repared in w a ter then its pH valu e adjusted to pH: 4.0.
G el filtration H PL C
The heterogenecity of polymers and proteins and the fraction compositions of the mixtures were estimated by the HPLC system (gel filtration chromatography). The system consists of Shimpack Diol 300 column (7.9mm ID x 500 mm) with Shimpack Precolumn Diol, Pump (LC-lOAi) and automatic sample injector (SIL-lOAi HPLC). The eluent was monitored at 280 and 254 nm by using UV detector (SPD-lOAi). A phosphate buffer containing 0.1 mol/L NaCl was used as a mobile phase at a flow rate of 1.0 mL/min at room temperature.
y-radiolysis
y-radiolysis of the 0.1% aqueous solutions of PAA, PNIPAAm, BSA, SOD and their mixtures PAA-BSA, PAA-SOD, PAA-Cu-BSA, PNIPAAm-BSA was performed by using a 60Co y-source (Picker 9 V). 5 mL sample solutions were irradiated at a position of 10 cm from the source. The dose rate was measured to be 40.2 Gy/h as determined by Fricke dosimetry9.
S p ectrop h otom etric M easu rem en ts
A Shimadzu UV-2401 PC spectrophotometer was used for spectroscopic analyses. The optical density changes were measured at 230 nm for PAA and PAA-Cu2+ and at 280 nm for BSA and PAA-Cu2 -BSA. The amount of Cu(I) occurred in solution after irradiation was determined spectrophotometrically10. 0.1 % Solution of neocuproine was freshly prepared in absolute ethanol. Cu(I) was determined by measuring the absorbance of its neocuproine complex at 457 nm.
F lu orescen ce M easu rem en ts
Fluorescence emission spectra were obtained using a spectrofluorimeter operating in quanta counting mode. The slits of excitation and emission monochromators were adjusted to 2 or 3 nm. The excitation was found to be at 280 nm. The fluorescence of proteins is widely used to study their behaviour depending on different influencing factors11121314. Protein concentration and volume were 0.71 mg/mL, 2 mL respectively.
3 . R E S U L T S A N D D I S C U S S I O N P A A -Cu2+ and P A A -C u 2+-B S A C om plexes
The formation of water soluble ternary complexes of PAA with BSA in the presence of divalent copper ions (Cu2+) was investigated. Under conditions where both polymer and protein have same (negative) charges and are incapable of binding to another in the absence of a mediator (metal ions), the divalent Cu2+ acts as a “fasteners” between BSA globules and PAA chains and promotes the formation of a soluble ternary complex which is stable under physiological conditions.
All the solutions were irradiated up to 1.2 kGy and the optical density changes versus radiation doses were plotted in Fig. 1.
PAA --- PAACu(Il)
— - A - BSA - * - PAA-Cu(U)-BSA — K— PAA BSA
Fig. 1. Optical Density Changes versus Radiation Doses
The percentage changes in optical density values of Cu2+ containing complexes do not change significantly up to the irradiation dose of 0.65 kGy. This probably indicate the stabilizing effect of Cu2+ added to polymer- protein. For further increase in irradiation dose it was observed that the decomposition rate of BSA and PAA- BSA increased sharply.
Aqueous solutions of PAA, PAA-Cu2+, BSA and PAA-Cu2+-BSA were also aerated or saturated with N2 and N20 before irradiation at 0.65 kGy dose. The changes occurred were measured by UV-Vis spectrophotometry and the percentage decomposition are presented in Table 1. The decomposition rates in all N20 saturated solutions were higher than aerated and N2 saturated solutions.
Table 1. Comparison of percentage decomposition of PAA and its complexes at various conditions
Medium PAA PAA-Cu^ BSA PAA-BSA PAA-Cu^-BSA
0 2 15.37 9.01 26.35 57.83 8.50
n2 10.81 3.31 18.41 25.52 2.23
n2o 30.15 9.12 56.76 61.05 6.59
[Cu2> 1.388x10 ^ M
The decomposition rate was observed to be minimum in solutions saturated with N2. This might be related to the presence of all radicals and molecules originally formed after irradiation of water without any conversion to each other9. Cu2+ ions may probably react with hydrated electrons and are reduced to Cu+ ions.
On the basis of spectrophotometric and HPLC results obtained, it is concluded that upon irradiation PAA undergoes degradation and also irradiation causes a change of the protein from the native form to the denaturated form2.
The reason for this seems to be tied in with the free radicals produced in water. Furthermore, it is noticed that upon irradiation, the rate of decomposition in PAA-BSA complexes is considerably higher than PAA-Cu2+
irradiated solutions of 0.1% PAA-Cu(II) and 0.2% PAA-Cu(II) by neocuproine showed that the concentration of Cu(I) increases with increasing radiation dose Fig.2.
Fig. 2. Effect of radiation dose on formation of Cu(I) in PAA-Cu(II) system [Cu2+] = 1.388x103 M, [PAA]=0.1 g/dL and 0.2 g/dL, nCu27 nAA=0.05
HPLC results2 of the irradiated and unirradiated PAA+Cu2++BSA mixtures at diferent irradiation doses, stable complexation of PAA with BSA via Cu2+ took place upon addition of copper ions into PAA-BSA mixture. It is remarkable that the character of the distribution of compounds in ternary mixture in contrast to PAA-BSA mixtures practically does not change during irradiation up to 1.2 kGy. At the high irradiation dose (2.5 kGy) the areas of the peak with low RT decreased and the distribution of compounds and the heterogenecity of solutions signifcantly difer from solutions irradiated at 1.2 kGy. This may cause the radiation-induced covalent crosslinking of particles. Therefore, the addition of Cu2+ ions to PAA-BSA mixture protects the PAA and BSA components of ternary PAA+Cu2++BSA complexes against radiation damage.
P A A -S O D System s
It is known that SOD, which is present in the cytosol of eukaryotic cells is a copper-zinc enzyme which catalyses the dismutation of the superoxide radical to hydrogen peroxide and molecular oxygen. The solutions of PAA, SOD and their equimolar mixture were irradiated and their HPLC results were compared to each other. The mixture of PAA-SOD up to 0.6 kGy irradiation dose was characterized in chromatograms by two peaks corresponding to free PAA and SOD2. The area of these peaks in the mixtures analogues to free polymer and enzyme systems did not change with increasing irradiation dose. It can be proposed that the higher capacity of SOD in complex formation with copper-zinc ions than PAA prevents the ternary PAA- metal-enzyme complex formation. This results demonstrated that SOD is a scavenger of superoxide radicals and prevents the covalent conjugate formation. SOD containing two Cu(II) and two Zn(II) atoms per molecule might take part in catalysis of the reaction and protect the macromolecules against radiation damage.
P oly (N IP A A m ) -B S A C om p lexes
The complex formation of poly (NIPAAm) with BSA was investigated by spectrofluorimetric measurements. The solutions of poly (NIPAAm) and BSA were irradiated at different doses with a 60Co y-source. The change of fluorescence intensity of polymer- protein conjugate with increasing irradiation dose was shown in Fig 3.
Log Gy
C
Fig. 3. The fluorescence intensity of poly(NIPAAm)-BSA conjugates at different radiation dose and temperature
It was observed that the fluorescence intensity of polymer- protein conjugate decreased with increasing irradiation dose and temperature. The fluorescence intensity showed a very little change for absorbed dose of up to 0.1 kGy, but it decreased considerably for doses higher than 0.1 kGy. We can propose that this phenomenon cause with the structural alteration of protein molecules in the composition of covalent conjugates. In order to obtain detailed information about structure, the change of maximum wavelength of conjugate with irradiation dose should be investigated.
4.CONCLUSIONS
Crosslinking and formation of water-soluble covalent conjugates of PAA and PNIPAAm with BSA molecules upon irradiation were observed in PAA-BSA and PNIPAAm-BSA mixtures. The addition of Cu2+ ions to PAA-BSA mixture protects the PAA and BSA components of ternary PAA-Cu2+-BSA(PEC.Cu2+) complexes against radiation damage.
PEC.Cu2+ + O2 PEC.Cu1+ + 0 2
Analogues protective effect was also observed in PAA-SOD systems. Cu(II) and Zn(II) ions in the structure of SOD molecules protect the free protein and protein component of PAA-SOD mixtures against radiation damage and prevent the PAA-SOD covalent conjugate formation. The mechanism underlying the protection effect might be related to the conversion of superoxide anion (0 2) to molecular oxygen (0 2) and hydrogen peroxide (H20 2) via Cu2+ ions. In the mixture of PAA-SOD, Cu-Zn-superoxide dismutase is the scavenger of the superoxide anion by the following equlibrium:
Me2+ - SOD + 2 0 2 + 2 H+ \4e+ - SOD + H20 2 + 0 2
Metal ions act as protective agents of the protein globules against radiation damage.
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