Aritalab:Lecture/Biochem/Extraction
From Metabolomics.JP
< Aritalab:Lecture | Biochem(Difference between revisions)
m |
|||
| Line 4: | Line 4: | ||
}} | }} | ||
</center> | </center> | ||
| − | =={{Bilingual| | + | =={{Bilingual|代謝物の性質|Metabolite Chemistry}}== |
| − | ;''Like dissolves like | + | {{Twocolumn| |
| + | Metabolites show huge chemical diversity demonstrating different characteristics. | ||
| + | * molecular weight and size | ||
| + | * polarity and volatility | ||
| + | * solubility and pK<sub>a</sub> (acid dissociation constant: [A-][H+]/[HA]) | ||
| + | | | ||
| + | 代謝物は幅広い化学的性質を持っています。 | ||
| + | * 分子量と分子の大きさ | ||
| + | * 極性と揮発度 | ||
| + | * 溶解度と酸解離定数 pK<sub>a</sub> ＝ [A-][H+]/[HA] | ||
| + | }} | ||
| + | |||
| + | ==={{Bilingual|極性|Polarity}}=== | ||
| + | {| class="wikitable" | ||
| + | |+ highly apolar ←———————————→ highly polar | ||
| + | |- | ||
| + | | lipids<br/>(fatty acids<br/>waxes<br/>terpenes) | ||
| + | | carotenoids<br/>chlorophylls<br/>steroids<br/>flavonoids | ||
| + | | phenolics<br/>alcohols | ||
| + | | amino acids<br/>organic acids<br/>organic amines<br/>alkaloids<br/>nucleosides | ||
| + | | sugars<br/>nucleotides<br/>phsphates<br/>metals<br/>salts | ||
| + | |} | ||
| + | |||
| + | :E.g. cyclohexane < benzene < chloroform < ether < acetone < ethanol < methanol < H2O | ||
| + | |||
| + | ==={{Bilingual|調節|Regulation}}=== | ||
| + | {{Twocolumn| | ||
| + | * substrate-level: cooperativity, feedback or feedforward control | ||
| + | * coenzymes: ATP, NAD, NADP, CoA, FMN, FAD, biotin, THF etc. | ||
| + | * allosteric: homoallostery (regulation by substrate), heteroallostery | ||
| + | * compartmentalization: source → sink flow, plastid, etc. | ||
| + | * hormone: growth factor, neurotransmitter, pheromone, etc. | ||
| + | * channeling: metabolon or multienzyme system | ||
| + | | | ||
| + | * 基質レベル: 共同性、フィードバック、フィードフォワード | ||
| + | * 補酵素: ATP, NAD, NADP, CoA, FMN, FAD, ビオチン, THF など | ||
| + | * アロステリック効果: ホモまたはヘテロなこうか | ||
| + | * 局在: ソース → シンク、色素体 | ||
| + | * ホルモン: 成長因子、神経伝達因子、フェロモンなど | ||
| + | * チャネリング: メタボロン、多酵素系 | ||
| + | }} | ||
| + | |||
| + | ==={{Bilingual|回転率|Turn-over rate}}=== | ||
| + | {| class="wikitable" | ||
| + | ! Metabolite || Turnover rate mM/s || species || Reference | ||
| + | |- | ||
| + | | glucose || 1.0 || ''S. cerevisiae'' aerobic on glucose | ||
| + | | De Koning & van Dam 1992 | ||
| + | |- | ||
| + | | glucose || 0.3 || Isolated adipocytes treated with insulin | ||
| + | | Marshall et al. 2004 | ||
| + | |- | ||
| + | | ATP || 1.5 | ||
| + | |rowspan="2"| ''S. cerevisiae'' aerobic on glucose (D=0.1/h) | ||
| + | |rowspan="2"| Rizzi et al. 1997 | ||
| + | |- | ||
| + | | ADP || 2.0 | ||
| + | |} | ||
| + | |||
| + | =={{Bilingual|抽出|Extraction}}== | ||
| + | |||
| + | ===Quenching=== | ||
| + | 代謝物を計測するには、酵素を失活させ、代謝物の分解を防がなくてはなりません。 | ||
| + | 光で分解する代表的物質は ''S''-アデノシル-L-メチオニン、酸化しやすい物質はリン酸化合物です。 | ||
| + | |||
| + | ;バクテリア | ||
| + | バクテリアの細胞膜は破れやすく、過塩素酸、熱エタノール、熱水、液体窒素はおろか冷メタノールでも破れてしまいます。 | ||
| + | 通常は、細胞膜を破壊して細胞内外の代謝物を一緒に計測したものから、培地だけを測ったものを差し引いて細胞内の濃度を算出します。 | ||
| + | |||
| + | ;酵母 | ||
| + | 冷メタノールを用いるのが一般的です。メタノール失活させる時間はできるだけ短くします。 | ||
| + | |||
| + | ;糸状菌 | ||
| + | 冷メタノールや液体窒素が一般的です。 | ||
| + | |||
| + | ;植物、動物 | ||
| + | 組織から目的の細胞を抽出する作業が最も大変です。通常は液体窒素を用いて凍らせ、すり鉢で粉砕します。 | ||
| + | |||
| + | ===Like dissolves like=== | ||
| + | |||
{{Twocolumn| | {{Twocolumn| | ||
Solvent for extraction is chosen to share similar polarity with the target compound. | Solvent for extraction is chosen to share similar polarity with the target compound. | ||
| Line 17: | Line 96: | ||
* 両方 <br/>→ メタノールなど | * 両方 <br/>→ メタノールなど | ||
}} | }} | ||
| − | |||
| − | |||
| − | =={{Bilingual|Separation & Purification | + | |
| + | |||
| + | =={{Bilingual|分離と精製|Separation & Purification}}== | ||
{{Twocolumn| | {{Twocolumn| | ||
Chromatography separates target compounds using their physico-chemical difference in adsorption or partition between stationary and mobile phases. | Chromatography separates target compounds using their physico-chemical difference in adsorption or partition between stationary and mobile phases. | ||
Latest revision as of 15:19, 2 June 2011
| Wiki Top | Up one level | レポートの書き方 | Arita Laboratory |
|
Natural product chemistry starts with "extraction" and "separation / purification."
[edit] Metabolite Chemistry
Metabolites show huge chemical diversity demonstrating different characteristics.
- molecular weight and size
- polarity and volatility
- solubility and pKa (acid dissociation constant: [A-][H+]/[HA])
[edit] Polarity
| lipids (fatty acids waxes terpenes) |
carotenoids chlorophylls steroids flavonoids |
phenolics alcohols |
amino acids organic acids organic amines alkaloids nucleosides |
sugars nucleotides phsphates metals salts |
- E.g. cyclohexane < benzene < chloroform < ether < acetone < ethanol < methanol < H2O
[edit] Regulation
- substrate-level: cooperativity, feedback or feedforward control
- coenzymes: ATP, NAD, NADP, CoA, FMN, FAD, biotin, THF etc.
- allosteric: homoallostery (regulation by substrate), heteroallostery
- compartmentalization: source → sink flow, plastid, etc.
- hormone: growth factor, neurotransmitter, pheromone, etc.
- channeling: metabolon or multienzyme system
[edit] Turn-over rate
| Metabolite | Turnover rate mM/s | species | Reference |
|---|---|---|---|
| glucose | 1.0 | S. cerevisiae aerobic on glucose | De Koning & van Dam 1992 |
| glucose | 0.3 | Isolated adipocytes treated with insulin | Marshall et al. 2004 |
| ATP | 1.5 | S. cerevisiae aerobic on glucose (D=0.1/h) | Rizzi et al. 1997 |
| ADP | 2.0 |
[edit] Extraction
[edit] Quenching
代謝物を計測するには、酵素を失活させ、代謝物の分解を防がなくてはなりません。 光で分解する代表的物質は S-アデノシル-L-メチオニン、酸化しやすい物質はリン酸化合物です。
- バクテリア
バクテリアの細胞膜は破れやすく、過塩素酸、熱エタノール、熱水、液体窒素はおろか冷メタノールでも破れてしまいます。 通常は、細胞膜を破壊して細胞内外の代謝物を一緒に計測したものから、培地だけを測ったものを差し引いて細胞内の濃度を算出します。
- 酵母
冷メタノールを用いるのが一般的です。メタノール失活させる時間はできるだけ短くします。
- 糸状菌
冷メタノールや液体窒素が一般的です。
- 植物、動物
組織から目的の細胞を抽出する作業が最も大変です。通常は液体窒素を用いて凍らせ、すり鉢で粉砕します。
[edit] Like dissolves like
Solvent for extraction is chosen to share similar polarity with the target compound.
- terpenoid, steroid (alicyclics and aromatics)
→ low polarity solvent (e.g. hexane) - saccharide, glycoside, amino acid
→ high polarity solvent (e.g. water) - both
→ methanol
[edit] Separation & Purification
Chromatography separates target compounds using their physico-chemical difference in adsorption or partition between stationary and mobile phases.
- normal phase
- polarity of stationary phase > polarity of mobile phase
- silica gel + organic solvent
- cyclohexane, benzene, chloroform, ether, acetone, ethanol, methanol
- reverse phase
- polarity of stationary phase < polarity of mobile phase
- coated silica beads + water
- water + acetonitrile, water + methanol, water + tetrahydrofuran (THF)
| (High Performance) Liquid Chromatography |
Gas Chromatography | |
|---|---|---|
| Adsorption-type 吸着型 |
Liquid-solid type (LSC)
silica gel, alumina, or porous polymers are used as adsorbent.
|
Gas-solid type (GSC)
Packed column has 2∼4 mm diameter and 30∼60 cm length, and is filled with diatomite, silica and silicon oil. |
| Partition-type 分配型 |
Liquid-liquid type (LLC) uses coated-silica beads.
|
Gas-liquid type (GLC) Capillary column has 0.2 mm diameter and > 25 m length, whose inside part is coated with silicon oil. |
- シリカへの吸着度合い
-COOH ＞ -OH > -NH2 > -C=O > -OCH3 > -Cl
