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Anal. Che" 1990, 62, 504-508
Coprecipitation of Trace Metals by DNA and RNA Molecules Kitao Fujiwara,* Ri-e Kojyo, and Kazuma Okada Faculty of Integrated Arts and Sciences, Hiroshima University, 1-1 -89 Higashisenda-machi, Hiroshima 730, Japan Yukio Kodama Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo 164, Japan Copredpila#on of trace metal lons In the aqueous sample was lnvestlgated by using several nuclelc acids (DNA and RNA) as the carrier. Afler the pH of the solutlon containing metal Ion was adjusted, DNA or RNA dissolved In NaOH solution was added. Precipltatton of DNA (or RNA) was done by the safflngoutmethod, addlngs"cMotMeand acetone tothe DNA-metal solution. Copreclpltatlon efflclencles for most posltlve metal lons were found to be maxlmum a1 pH 2-3 when sodlum hydroxide-acetate buffer was used. Negatlve lons such as Cr20,1-, Mo7O2,@-,and PtCIt- were not preclpRated wlth DNA In the pH range from 1 to 12. It Is also found that copreclpltatlons of cobalt( I I I ) complexes were dominated by thelr charge, Le., the complexes havlng plus three charge are copredpltated wlth DNA at the hlgh rate but the complexes of zero or mlmnr charge are not. The antlcancer drug cls-platln Is negllglbly copreclpltated wlth DNA. The present copreclpltatlon method also allows the preconcentratlon of trace metal lons except Iron, calclum, and magnesium, whlch were lntrlnsically contalned In DNA and RNA at the apprectable concentratlon.
When trace metal distribution was measured in natural waters, preconcentration and isolation of analyte from the sample matrices are required; their concentration is lower than the detection limit and appreciable interferences of coexistent species sometimes appear in the measurement methods. Solvent extraction and coprecipitation methods were, therefore, reported as the sample pretreatment for analyzing the trace metals in natural waters. Hydroxides of metal ions such as Al(OH), and Fe(OH), are the common coprecipitator (1-3). Also, gallium (4), indium (5,6),magnesium (7), hafnium (8), and zirconium (9) hydoxides are utilized as the coprecipitator for multielements detection in aqueous samples by inductively coupled plasma (ICP) atomic emission spectrometry. Other than the hydroxide formation of metal ions, neodymium fluoride (10) was used for collection of actinide ions. Also, the complex formation between cobalt and l-pyrrolidinecarboditioate (Co-APDC) is investigated as coprecipitation techniques for seawater analysis for Ni, Cu, Cd ( l l ) Cr , (12), V, and Mo (13, 14). In the present paper, a new coprecipitation method using biological materials such as nucleic acids is investigated for collection of trace metal ions in the aqueous samples. I t is known that several transition metals combine with DNA and RNA and increase their stabilities. Also, zinc and magnesium participate with the transformation of nucleic acids (15, 16). Recently, some DNA and RNA molecules are commercially available a t a rather inexpensive price, and here, we would like to evaluate these species as coprecipitating agents for trace metal ions in aqueous samples in terms of elucidation of interaction between trace metal ions and nucleic acids in natural waters. EXPERIMENTAL SECTION Reagents. Deoxyribonucleic acids extracted from salmon sperm purchased from Wako Pure Chem. Ind., Ltd. (047-17322), herring sperm purchased from Sigma Chemical Co. (D-3159),and deoxyribonucleic acid sodium salt from salmon testes (Type 111)
purchased from Sigma (D-1626)were used as the coprecipitation agents of DNA. ribonucleic acid from Tolula yeast purchased from Sigma (R-6625) and ribonucleic acid sodium salt from yeast purchased from Kojin Co., Ltd. (1600), were used as RNA. The standard solutions of metal ions measured were as follows: (Co) CoCl, (lo00 pg of Co/mL) in 1mol/L HC1; (Cu) CuCl, (lo00 bg of Cu/mL) in 0.1 mol/L HCl; (Cd) CdCl, (100 pg of Cd/mL) in 1mol/L HCI; (Mn) MnC12 (lo00 pg of Mn/mL) in 0.02 N HC1; (Ni) NiC1, (1000 pg of Ni/mL) in 0.1 N HCI; (Pt)H2PtC1, (lo00 bg of Pt/mL) in 1 mol/L HC1, which were purchased from Wako. Chromium(II1) nitrate (Cr(N03)3.6H20) at analytical grade purchased from Kanto Chemical Co. and spectroscopy grade potassium dichromate purchased from Katayama Chemical Co. were dissolved in the aqueous solution and used as the chromium standard solution. Ammonium molybdate ((NH4)6Mq0u.4H20) purchased from Katayama was dissolved in aqueous solution and was used as the molybdenum standard. The following cobalt(II1) complexes synthesized by Dr. Katsuhiko Miyoshi, Hiroshima University, were also investigated in their ability to absorb to nucleic acids: [Co(NH3)&13, [Co(NH3)&1]Cl2,[Co(sep)lCl3, [Co(dien)C121C104, [C~(en)~lCl~, K[Co(C204)(gly)21,Na[Co(edta)l. cis-Platin (cis-[Pt(NH,),Cl,]), anticancer drug, was a gift from Nippon Kayaku Co. Ltd. Abbreviations of ligands are as follows: en = ethylenediamine, dien = diethylenetriamine, and CH2NHCHZCH2NHCHz
\ / ~ e =p N-CH2NHCHzCHZNHCHZ-N 'CH,NHCH,CH,NHCH, /
Apparatus. Atomic absorption spectrometry was done with a Perkin-Elmer Model 603 atomic absorption spectrophotometer for flame atomic absorption spectroscopy (AAS)and a Shimadzu Model AA-640-13 atomic absorption spectrophotometer with a graphite furnace controller, Model GFA-4, and an autosample injector, Models ASG-1 and AIU-1, for graphite furnace AAS. pH dependence of coprecipitation efficiencies of the metal ions was determined by the air-acetylene flame AAS. Metal amounts (28 elements) intrinsically contained in DNA and RNA used in the present study were surveyed by a Seiko Model JY-48 ICP atomic emission spectrometer. A centrifuge made by Kubota, Model KN-70, was used in the separation of precipitates. Procedure. To calculate the collection efficiency of metal ions in the coprecipitation with DNA and RNA, the following procedure was performed: Metal ions were diluted into a buffer solution of 1mol/L sodium acetate and 1mol/L acetic acid. After the pH was adjusted, volume of solution was made to 3 mL. A 0.5-mL portion of DNA or RNA solution (2% (w/v)) was then added, where DNA or RNA was made by dissolving them into 10 mmol/L NaOH solution. The mixed solution was left for at least 30 min at 1-5 "C, and 0.5 mL of 2 mol/L NaCl and 6 mL of acetone were added to the solution in this order. The white precipitate appears when acetone is added. After this solution was allowed to stand for 30 min, the precipitate was collected by centrifugation (2000 rpm, 15 min). The centrifugal rotor was a swing type and tubes made of glass were used for this centrifugation. After the supernatant was discarded, the centrifugal tube with the precipitate was rinsed with ethanol once. The precipitate was dissolved into a mixture of 0.5 mol/L ammonia water and 0.2 mol/L ethylenediaminetetraacetic acid diammonium salt ((NH,),-EDTA). RESULTS AND DISCUSSION Optimization of Coprecipitation Procedure. Several ways are known for the precipitation of DNA or RNA from their solutions. Denaturation by the strong acids such as
0003-2700/90/0362-0504$02.50/0 0 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 5, MARCH 1, 1990
505
Table I. Contents of Metals in Nucleic Acids Used in this Study, p g / $
As Ag
A1 Au
B Ba Ca Cd Cr
cu Fe
Ga Ge Mg Mn Mo Ni
Pb Sb
Si Sn Sr Ti V Y Zr
Zn
AHW(1)
AHW(2)
AHW(3)
AHW(4)
AHW(5)
D-3159
D-1625
R-6625
K-1600
11
