Anal. Chem. 2004, 76, 233-237
Technical Notes
Enzymatic Probe Sonication: Enhancement of Protease-Catalyzed Hydrolysis of Selenium Bound to Proteins in Yeast J. L. Capelo,*,†,§ P. Xime´nez-Embu´n,‡ Y. Madrid-Albarra´n,‡ and C. Ca´mara*,‡
Departamento de Engenharia Quı´mica, Instituto Superior Te´ cnico de Lisboa, Avda Rovisco Pais, s/n, 1049-001, Lisboa, Portugal, and Departamento de Quı´mica Analı´tica, Facultad de Quı´micas, Universidad Complutense de Madrid, Avda Complutense, s/n E-28040 Madrid, Spain
This paper describes the dramatic activity enhancement of two proteolytic enzymes (protease XIV and subtilisin) when treated with an ultrasonic probe and their application to total Se determination and Se speciation in biological samples. Total Se extraction from enriched yeast is performed with 10 mg of yeast plus 1 mg of protease with a sonication time of 5 s, whereas 30 s is needed for extracting selenomethionine. In both cases, aqueous media was used. This spectacular finding is important because the enzymatic procedure usually requires a long treatment period at 37 °C. In addition to this major advantage, no control temperature is needed and the risk of species interconversion is drastically reduced or inhibited (the same Se species were detected after different sonication times). Moreover, the extraction is performed in water, minimizing contamination risk and without further pH adjustment. The new sample treatment proposed has been successfully applied to selenium speciation in yeast using chromatographic separation (HPLC) coupled to inductively coupled plasma-mass spectrometry. Interest in the determination of selenium and its species in biological samples has steadily increased in recent years.1,2 Selenium is a necessary component at the active site of glutathione peroxidase, the enzyme that controls the reduction of peroxides and hydroperoxides, slowing down the aging process.3-5 In addition, it is recognized that selenium-enriched products provide * Corresponding authors. E-mail:
[email protected]; phone: 34 91 394 43 18; fax: 34 91 394 43 29. E-mail:
[email protected]. † Instituto Superior Te´cnico de Lisboa. ‡ Universidad Complutense de Madrid. § Current address: Universidade Tecnica de Lisboa, Instituto Superior Tecnico de Lisboa, Torre Sul 11 Amdar, Avda Rovisco Pais s/n, 1049-001 Lisboa, Portugal. Phone/Fax: 351-21841-9596. (1) Birringer, M.; Pilawa, S.; Flohe´, L. Nat. Prod. Rep. 2002, 19, 693-718. (2) Stadtmen, T. C. J. Biol. Chem. 1991, 266, 257-262. (3) Pyrzynska, K. Microchim. Acta 2002, 140, 55-62. (4) Lobinski, R.; Edmonds, J. S.; Suzuki, K. T.; Uden, P. C. Pure Appl. Chem. 2000, 72 (3), 447-461. (5) De la Calle Guntin ˜as, M. B.; Bordin, G.; Rodrı´guez, A. R. Anal. Bioanal. Chem. 2002, 374, 369-378. 10.1021/ac034871d CCC: $27.50 Published on Web 11/25/2003
© 2004 American Chemical Society
protection against various forms of cancer.6 Thus, the interest in selenium speciation in food supplements (such as seleniumenriched yeast) and biological samples is now increasing, and many papers on new selenium speciation methodologies have been published in the last 10 years.7-10 One of the main problems cited by different authors is directly related to the extraction methodology for quantitative recovery of selenium species. Usually selenium in biological samples is bound to proteins and peptides, with the consequent need for cleavage of these structures before analysis. Different approaches such as enzymatic and basic hydrolysis have been proposed, the former being the most widely used. However, the process sometimes requires two consecutive steps, each of 24 h at 37 °C.11 This methodology is extremely timeconsuming, the efficiency of the process is far from being quantitative, and the risk of selenium interconversion is very high. All these drawbacks probably explain why selenium speciation results are hardly comparable when this methodology is applied by different authors in intercomparison exercises. Ultrasonic liquid extraction (USLE) has been applied for total selenium studies in combination with either ultrasonic bath or ultrasonic probe under different acid conditions.12-14 Table 1 summarizes the state of art concerning sample treatment using USLE for total selenium determination and Se speciation from several samples. Taking into account that the ultrasonic probe (6) Clark, L. C.; Combs, G. F., Jr.; Turnbull, B. W.; Slate, E. H.; Chalker, D. K.; Chow, J.; Davis, L. S.; Glover, R. A.; Graham, G. F.; Gross, E. G.; Krongrad, A.; Lesher, J. L., Jr.; Park, H. K.; Sanders,, B. B., Jr.; Smith, C. L.; Taylor, J. R. J. Am. Med. Assoc. 1996, 276, 1957-1963. (7) Chassaigne, H.; Che´ry, C. C.; Bordin, G.; Rodrı´guez, A. R. J. Chromatogr.. A 2002, 976, 409-422. (8) Ruiz Encinar, J.; Ruzik, R.; Buchmann, W.; Tortajada, J.; Lobinski, R.; Szpunar, J. Analyst 2003, 128, 220-224. (9) B’Hymer, C.; Caruso, J. A. J. Anal. At. Spectrom. 2000, 15, 1531-1539. (10) Sutton, K. L.; Ponce de Leo´n, C. A. S.; Ackley, K. L.; Sutton, R. M. C.; Stalcup, A. M.; Caruso, J. A. Analyst 2000, 125, 281-286. (11) Moreno, P.; Quijano, M. A.; Gutie´rrez, A. M.; Pe´rez-Conde, M. C.; Ca´mara, C. J. Anal. At. Spectrom. 2001, 16, 1044-1050. (12) Bermejo-Barrera, P.; Mun ˜iz-Naveiro, O.; Moreda-Pin ˜eiro, A.; BermejoBarrera, A. Anal. Chim. Acta 2001, 439, 211-227. (13) El Azouzi, H.; Cervera, M. L.; de la Guardia, M. J. Anal. At. Spectrom. 1998, 13, 533-538. (14) Me´ndez, H.; Alava, F.; Lavilla, I.; Bendicho, C. Anal. Chim. Acta 2002, 452, 217-222.
Analytical Chemistry, Vol. 76, No. 1, January 1, 2004 233
Table 1. Total Se and Se Species Recovery from Different Samples Using USLE in the Sample Treatment aim
sample
treatment
Se recovery (%)
ref
total Se total Se Se speciation
seafood seafood White Clover CRM 402 yeast
Se sequential extraction
yeast
100 100 28.5 ( 3.5 36.7 ( 0.3 47.6 ( 5.5 13 10 15.2 ( 1.0
12 14 21
Se speciation
ultrasonic bath, 35 min, 90 °C, 2.5 M HNO3 probe sonication, 3 min, 50% amplitude, 0.5 M HNO3 ultrasonic bath, 2 × 30 min, methanol/water (1+1) ultrasonic bath, 2 × 30 min, methanol/water (1+1) + 0.28 M HCl ultrasonic bath, 2 × 30 min, methanol/water (1+1) + 4% NH3 ultrasonic bath, 1 h, methanol/water (1+9) + 0.2 M HCl ultrasonic bath, 1 h, 30 mM Tris-HCl buffer (pH 7) + 0.1 mM PMSF probe sonication, 10 × 5 s, 10 mM Tris HCl buffer (pH 8)
provides 100 times more energy than the ultrasonic bath,15 creating conditions far from mild, the last one seems to be, in a first approach, the adequate ultrasonic device that should be used for USLE, especially when species preservation is the main analytical task. Total Se recovery using an ultrasonic bath requires a treatment time longer than 30 min, high acid concentration, and high temperature.12 The use of an ultrasonic probe allows the reduction of the treatment time, but a high acid concentration is still required.14 Unfortunately, these conditions are incompatible with the integrity of the Se species. This is probably why, to the best of the author’s knowledge, ultrasonic probes have never been used in studies on Se speciation. The use of enzymes in conjunction with ultrasonic energy has already been reported,16,17 and it seems that the activity of the enzyme is enhanced. In this paper, we describe the use of focused ultrasound in conjunction with enzymatic hydrolysis, enzymatic probe sonication EPS, for total selenium and selenomethionine determination in a selenium-enriched yeast sample. The optimization of those parameters affecting the process such as extraction media, sample/enzyme ratio, and sonication time was described. EXPERIMENTAL SECTION Apparatus. A Sonopuls ultrasonic homogenizer (Bandelin) fitted with a HF generator 2200 was used to extract the samples. The homogenizer was equipped with a titanium microtip of 3-mm diameter, and the power was set to 20 W. The frequency was fixed at 20 kHz. A HP-4500 (Tokyo, Japan) inductively coupled plasma-mass spectrometer (ICPMS) fitted with a Babington nebulizer and a Scott double-pass spray chamber cooled by a Peltier system was used for total selenium determination. When separation of the species was required, the ICPMS was coupled with the HPLC chromatographic system. The connection was made with a polyether ether ketone (PEEK) tubing. For chromatographic experiments, a Jasco PU-2089 Plus HPLC pump (Tokyo, Japan) was used as the sample delivery system under isocratic conditions. The pump was fitted with a six-port sample injection valve (model 7725i, Rheodyne, Rohner Park, CA) including a 200-µL injection loop. The separation was performed on a 250 × 4.1 mm i.d., 10-µm Hamilton PRP-X200 cationic exchange (Hamilton, Reno, NV) column. Se species quantification was performed in the peak area mode. Data evaluation was carried out with HP ChemStation software. (15) Mason, T. J. Sonochemistry; Oxford University Press: New York, 1999. (16) Oulahal-Lagsir, N.; Martial-Gros, A.; Bonneau, M.; Blum, L. J. Biofouling 2003, 19, 159-168. (17) Barton, S.; Bullock, C.; Weir, D. Enzyme Microb. Technol. 1996, 18, 190194.
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Reagents and Standards. All reagents were of analytical grade and were used without further purification. For the acidic extraction, hydrochloric acid was prepared by diluting the appropriate volume of concentrated HCl (Merck). HPLC-grade methanol (SDS, Barcelona, Spain) was used for extraction in alcoholic solution, pure or appropriately diluted. For the enzymatic extraction, Tris-HCl buffer (pH 7.5) (Fluka) and the nonspecific proteases Streptomyces griseus (protease XIV) and Bacillus subtilisin (subtilisin), (Sigma-Aldrich, St. Louis, MO) were used. Selenomethionine (SeMet) and selenocystine (SeCys2) (SigmaAldrich, St. Louis, MO) were prepared in doubly deionized Milli-Q water (Millipore, Bedford, MA). Hydrochloric acid (3%) was added to dissolve SeCys2. Inorganic selenium solutions were obtained by dissolving sodium selenite and sodium selenate (Merck, Darmstadt, Germany) in Milli-Q water. The stock solutions containing 1000 mg L-1 selenium from each compound were stored in dark at 4 °C. Working solutions were prepared daily by appropriate dilution. Before injecting into the chromatographic system, each sample was filtered using a syringe-driven filter unit containing a 0.45-µm cellulose filter (PVDF Durapore, Millipore). For HPLC-ICPMS studies, the mobile phase was 4 mM pyridine formate in 3% methanol. The eluent was prepared by diluting commercial pyridine (Merck) with distilled water and adjusting the pH to 2.8 with HCl. A lyophilized selenium-enriched yeast was used for these studies. The SelenoPrecise yeast was industrially produced by Pharma Nord (Vejle, Denmark), and a total selenium concentration of 1300 mg kg-1 is the value given by the manufacturer. Other materials were used to assess the extraction efficiency such as mussel (CRM 278), certified for total selenium content, and a lyophilized oyster sample (BCR 710) with a recommended value in Se content. Procedures. Total Selenium Determination. A 10-mg sample of yeast was placed in a 1.5-mL Eppendorff, and ∼1 mg of the nonspecific protease S. griseus (protease XIV) was added in 1 mL of Milli-Q water. The mixture was sonicated during 5 s at 20 W of power intensity. The resulting suspension was centrifugated, and 20 µL of the liquid phase was diluted to 10 mL with distilled water. The total selenium content of the sample was measured by ICPMS. For this purpose, the isotopes 78Se and 82Se were monitorized. The negligible differences between Se concentration using both isotopes for Se quantification led us to confirm the absence of interferences. 103Rh was used as an internal standard. Selenium Species Determination. Selenium species extraction was carried out following enzymatic probe sonication (EPS) conditions. The extraction was performed in 1 mL of distilled water, where ∼10 mg of yeast and 1 mg of the enzyme were
Table 2. Experimental Conditions for Se Determination by HPLC-ICPMS
analytical column eluent eluent flow rate elution program injection volume
HPLC Parameters PRP-X200 4 mM pyridine formate solution, pH 2.8, 97 mL H2O + 3 mL of methanol 1 mL min-1 isocratic 200 µL
ICPMS Conditions for HP-4500 Instrument forward power 1350 W plasma gas (Ar) flow rate 15 L min-1 auxiliary gas (Ar) flow rate 1.3 L min-1 carrier gas (Ar) flow rate 1.1 L min-1 nebulizer type Babington spray chamber type Scott double-pass data acquisition mode time-resolved analysis 78Se, 82Se monitorized isotopes
sonicated for 30 s. The resulting suspension was passed through a 0.45-µm filter and subsequently diluted with Milli-Q water. Selenium species were quantified at isotopes 78Se and 82Se by the standard addition procedure using HPLC-ICPMS. The instrumental conditions are summarized in Table 2. RESULTS AND DISCUSSION SelenoPrecise yeast, provided by Pharma Nord, was selected as a representative yeast sample because it has been thoroughly analyzed by the scientific community and the homogeneity and stability of the species (mainly SeMet) has been demonstrated through an intercomparison exercise.18 Total Selenium Determination. Preliminary studies were performed to evaluate the influence of the medium composition and sonication time on selenium extraction with the ultrasonic probe. Different sample mass/volume ratios were also investigated. In these experiments, yeast was used as the target sample. Table 3 shows that when common extractants were used with the sonication probe, the maximum recovery achieved was not higher than 30% of the total selenium content, estimated value 1300 mg kg-1. Neither acidification of the media nor the use of longer sonication times resulted in a better extraction efficiency. When sonication time was higher than 90 s, the suspension was introduced in an ice bath in order to avoid the effectiveness of ultrasonic irradiation with increasing temperature. However, sonication times ∼90 s are not long enough to increase the solution temperature to undesired levels. The lowest Se recoveries (
