Metalloporphyrin

原卟啉IX是血红素和叶绿素生物合成途径之间的最后一个常见中间体。镁的加入将这种分子导向叶绿素的生物合成。分支点下游的第一步由镁螯合酶催化，是一个高度调节的过程。相应的产物镁原卟啉IX被认为在质体到核的通讯中起着重要的信号分子作用。为了获得更多关于叶绿素生物合成途径和镁原卟啉IX衍生物功能的信息，我们在拟南芥中鉴定了一种镁原卟啉-IX甲基转移酶（CHLM）敲除突变体，该突变诱导镁原卟啉Ⅸ下游的阻断和这种叶绿素生物合成中间体的积累。我们的研究结果表明，CHLM基因对叶绿素的形成至关重要，随后对光系统I和II以及细胞色素b6f复合物的形成也至关重要。如前所述，对chlm突变体基因表达的分析提供了一个独立的指示，表明镁原卟啉IX是核光合基因表达的负效应器。此外，这表明CHLM的产物镁原卟啉IX甲酯可能参与叶绿体到细胞核的信号传导。最后，在chlm突变体中检测到镁螯合酶CHLH亚基水平的转录后上调，这很可能对应于该蛋白在质体内的特异性积累。这一结果表明，当叶绿素生物合成途径在这一特定步骤中断时，CHLH亚基可能起着重要的调节作用[2]。

四吡咯中间体分析[2]
在黑暗中，将约20 mg冷冻叶材料在500μL丙酮中均质化：0.125M NH4OH（9:1，v/v），并在4°C下离心。上清液用1mL研磨介质稀释，用1mL己烷提取3次。在完全消除己烷相后，丙酮相直接用于荧光测量或在氩气下干燥，然后溶解在甲醇中：5 mM磷酸四丁基铵（70:30 v/v）用于HPLC分析。在室温下，用Biologic的荧光分光光度计MOS-450记录570至690 nm的荧光发射光谱，激发波长为402 nm，用于检测原卟啉IX，或416 nm，用于探测镁原卟啉IX和Mg原卟啉IX甲酯，或440 nm，用于检测叶绿酸。HPLC分析如所述，不同之处在于通过在420 nm处的吸光度检测和荧光检测（λ激发420 nm/λ发射595 nm或λ激发420 nm/λ发射625 nm）监测洗脱。使用如（6）中所述制备和鉴定的原卟啉IX、镁原卟啉IX、镁原卟啉Ⅸ甲酯标准品。需要时，在提取前，将植物与10 mM ALA和5 mM MgCl2在10 mM Hepes pH 7.0中孵育过夜。

[1]. GRANICK S. Magnesium protoporphyrin as a precursor of chlorophyll in Chlorella. J Biol Chem. 1948;175(1):333-342.

[2]. Knock-out of the magnesium protoporphyrin IX methyltransferase gene in Arabidopsis. Effects on chloroplast development and on chloroplast-to-nucleus signaling. J Biol Chem. 2007;282(4):2297-2304.

Modification of expression of nuclear encoded photosynthetic genes [2]
On the opposite, the level of LHCB mRNAs is greatly decreased in the mutant, due to transcriptional regulation. Since the mutant specifically accumulates Mg protoporphyrin IX without any treatment, our result provides an independent indication that Mg protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously suggested in Norfluorazon treated plants. Moreover, the chlm mutant behaves like a super-repressor of the LHCB promoter and seems more efficient in repressing LHCB expression than plants treated with Norflurazon. The extent of repression may be due to a different level of accumulation of Mg protoporphyrin IX in the plant. A second possibility may be linked to the complete absence of Mg protoporphyrin IX methylester and its derivatives in the mutant. This suggests that one of these components acts as a positive effector of nuclear photosynthetic gene expression. Mg protoporphyrin methylester itself may be a positive effector. In support of this latter hypothesis, have shown that, in the absence of Norflurazon, the barley xantha l mutant that is defective in the Mg protoporphyrin methylester cyclase, accumulates Mg protoporphyrin IX methylester and has a high level of LHCB expression. In addition, reported positive correlation between LHCB expression and methyltransferase activity in tobacco CHLM antisense and sense RNA mutants.
The activity of Mg protoporphyrin IX methyltransferase is obviously dependent on the availability of Mg protoporphyrin IX but is also certainly adjusted to levels of Ado-Met and Ado-Hcy in the chloroplast (see for instance 38). The differential effects of Mg protoporphyrin IX and Mg protoporphyrin IX methylester on LHCB expression would consequently finely attune the synthesis of light harvesting complexes to the one-carbon metabolism.

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Increase of CHLH level [2]
In the chlm mutant we observed a strong increase in the level of mature CHLH, whereas that of CHLH mRNA was slightly reduced. Our data indicate that stabilization of the protein inside chloroplasts probably occurs in the chlm mutant. One possible mechanistic explanation for this accumulation could be traced to the increased level of Mg protoporphyrin IX in this mutant. Indeed, a tight channeling of substrate between the Mg chelatase and the methyltransferase has been suspected for a long time due to the absence of detectable Mg protoporphyrin intermediates in standard conditions. More recently, the physical interaction between CHLM and CHLH has been shown. During diurnal growth, the Mg chelatase activity peaks during the transfer from dark to light while the methyltransferase activity maximum follows only a few hours later. During this period, the Mg protoporphyrin IX level is transiently higher than that of Mg protoporphyrin IX methylester. It is possible that Mg protophorphyrin IX binds to CHLH, preventing the transitory accumulation of free Mg protoporphyrin IX when CHLM is not sufficiently present/active. This could both stabilize the rapidly turning over CHLH protein nd protect it against major photooxidative damage generated by free Mg protoporphyrin IX. Whatever the mechanism underlying CHLH accumulation, one can question whether its concomitant increase with Mg protoporphyrin IX plays a role in mediating the plastid-to-nucleus regulatory pathway.[2]

C34H32MGN4O4

584.95

584.227

14947-11-6

Mn(II) protoporphyrin IX;21393-64-6;Cu(II) protoporphyrin IX;14494-37-2;Ni(II) protoporphyrin IX;15415-30-2;Ga(III) protoporphyrin IX;222556-71-0;Cd(II) protoporphyrin IX;80216-25-7;Pt(II) protoporphyrin IX;98303-94-7

167213

Typically exists as solid at room temperature

3.612

90.48

2

8

6

43

995

0

O=C([O-])CCC1C2N3[Mg+2]45[N-]6C(=C(CCC(=O)[O-])C(C)=C6C=C7N4=C(C(C)=C7C=C)C=C8[N-]5C(C(C)=C8C=C)=CC=3C=1C)C=2

REJJDEGSUOCEEW-UHFFFAOYSA-L

InChI=1S/C34H34N4O4.Mg/c1-7-21-17(3)25-13-26-19(5)23(9-11-33(39)40)31(37-26)16-32-24(10-12-34(41)42)20(6)28(38-32)15-30-22(8-2)18(4)27(36-30)14-29(21)35-25;/h7-8,13-16H,1-2,9-12H2,3-6H3,(H4,35,36,37,38,39,40,41,42);/q;+2/p-2

magnesium;3-[18-(2-carboxylatoethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethylporphyrin-21,24-diid-2-yl]propanoate;hydron

14947-11-6; Magnesium protoporphyrin; Mg(II) protoporphyrin IX; Mgproto; PROTOPORPHYRIN IX CONTAINING MG; Mg Protoporphyrin; Divinyl-Mg-protoporphyrin; magnesium;3-[18-(2-carboxylatoethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethylporphyrin-21,24-diid-2-yl]propanoate;hydron;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
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