• 분석과학
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• 제26권6호
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• pp.357-363
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• 2013

Selenium-containing proteins were separated from selenium-enriched yeast (SEY) using Trizol$^{(R)}$ reagent followed by anion exchange (AE) chromatography. This method is simpler and less time consuming than electrophoresis. Five selenium containing proteins were identified by on-line AE HPLC-ICP/MS (high performance liquid chromatography-inductively coupled plasma/mass spectrometry). Each protein was enzymatically hydrolyzed to seleno-amino acids and separated with RP (reverse phase) HPLC for the identification of selenoproteins.

• Bulletin of the Korean Chemical Society
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• 제34권12호
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• pp.3817-3824
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• 2013

Various separation modes in HPLC, such as anion exchange (AE), reversed-phase (RP), and affinity (AF) chromatography were examined for the separation of selenium species in human blood serum and urine. While RP- and AE-HPLC were mainly used for the separation of small molecular selenium species, double column AF-HPLC achieved the separation of selenoproteins in blood serum efficiently. Further, the effluent of AF-HPLC was enzymatically hydrolyzed and then analyzed with RP HPLC for selenoamino acid study. The versatility of the hybrid technique makes the in-depth study of selenium species possible. For quantification, post column isotope dilution (ID) with $^{78}Se$ spike was performed. ORC ICP/MS (octapole reaction cell inductively coupled plasma/mass spectrometry) was used with 4 mL $min^{-1}$ Hydrogen as reaction gas. In urine sample, inorganic selenium and SeCys were identified. In blood serum, selenoproteins GPx, SelP and SeAlb were detected and quantified. The concentration for GPx, SelP and SeAlb was $22.8{\pm}3.4\;ng\;g^{-1}$, $45.2{\pm}1.7\;ng\;g^{-1}$, and $16.1{\pm}2.2\;ng\;g^{-1}$, respectively when $^{80}Se/^{78}Se$ was used. The sum of these selenoproteins ($84.1{\pm}4.4\;ng\;g^{-1}$) agrees well with the total selenium concentration measured with the ID method of $87.0{\pm}3.0\;ng\;g^{-1}$. Enzymatic hydrolysis of each selenium proteins revealed that SeCys is the major amino acid for all three proteins and SeMet is contained in SeAlb only.

• 분석과학
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• 제27권2호
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• pp.92-99
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• 2014

본 연구에서는 음이온 고체상 추출법 (AE SPE; anion exchange solid phase extraction)을 사용하여 간섭요인을 제거한 후, 친화 크로마토그래피 AF HPLC; affinity high performance liquid chromatography)와 유도결합 플라스마 질량분석법 (ICP/MS; inductively coupled plasma/mass spectrometry)을 사용하여 한국인의 혈청에서의 셀레노 단백질을 분리하고 정량하였다. 먼저 동위원소 희석법으로 셀레늄 총량을 분석한 결과, 건강한 한국 사람의 혈청내 평균농도는 $94.3{\pm}2.3ngg^{-1}$ 이었다. AE SPE와 AF 컬럼을 online으로 연결하여 셀레노단백질인 glutathione peroxidase (GPx), selenoprotein P (SelP), selenoalbumin (SeAlb)을 분리하고, 후 컬럼 동위원소 희석법 (PC ID; post column isotope dilution)으로 정량하였다. 혈청 인증표준물인 BCR-637을 분석한 결과 전체 셀레노 단백질의 합은 $85.4{\pm}3.4ngg^{-1}$으로 문헌값인 $81{\pm}7ngg^{-1}$과 일치하는 결과를 얻을 수 있었다. 20 명의 건강한 한국인의 혈청에서 얻은 셀레노 단백질 GPx, SelP 및 SeAlb 의 농도는 각각 $12.1{\pm}1.4ngg^{-1}$, $57.2{\pm}2.0ngg^{-1}$, 그리고 $20.0{\pm}1.9ngg^{-1}$ 이었으며 이들의 합인 $89.3ngg^{-1}$은 셀레늄의 총량값인 $94.3ngg^{-1}$과 거의 같은 값으로 혈청에서의 셀레늄은 주로 셀레노 단백질로 구성되어 있음을 알았다. 이 중 GPx의 농도는 간섭을 제거하기 전인 $25.0ngg^{-1}$에 비해 50% 이상 크게 감소하였는데 이로서 간섭은 주로 GPx에 포함되어 있음을 확인할 수 있었다. AE SPE는 간섭요인인 Cl과 Br을 제거 하는데 매우 효과적임을 보여주었다.