Metalloporphyrin

Protoporphyrin IX is the last common intermediate between the heme and chlorophyll biosynthesis pathways. The addition of magnesium directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the magnesium chelatase and is a highly regulated process. The corresponding product, magnesium protoporphyrin IX, has been proposed to play an important role as a signaling molecule implicated in plastid-to-nucleus communication. To get more information on the chlorophyll biosynthesis pathway and on magnesium protoporphyrin IX derivative functions, we have identified an magnesium protoporphyrin IX methyltransferase (CHLM) knock-out mutant in Arabidopsis in which the mutation induces a blockage downstream from magnesium protoporphyrin IX and an accumulation of this chlorophyll biosynthesis intermediate. Our results demonstrate that the CHLM gene is essential for the formation of chlorophyll and subsequently for the formation of photosystems I and II and cytochrome b6f complexes. Analysis of gene expression in the chlm mutant provides an independent indication that magnesium protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously reported. Moreover, it suggests the possible implication of magnesium protoporphyrin IX methyl ester, the product of CHLM, in chloroplast-to-nucleus signaling. Finally, post-transcriptional up-regulation of the level of the CHLH subunit of the magnesium chelatase has been detected in the chlm mutant and most likely corresponds to specific accumulation of this protein inside plastids. This result suggests that the CHLH subunit might play an important regulatory role when the chlorophyll biosynthetic pathway is disrupted at this particular step [2].

Tetrapyrole intermediate analysis [2]
Approximately 20 mg of frozen leaf material was homogenised in 500 μL acetone: 0.125M NH4OH (9:1, v/v) in the dark and centrifuged at 4°C. The supernatant was diluted with 1 mL grinding medium and extracted 3 times with 1 mL hexane. After the complete elimination of the hexane phase, the acetone phase was either directly used for fluorescence measurement or dried under argon before solubilization in methanol: 5 mM tetrabutylammonium phosphate (70:30 v/v) for HPLC analysis. Florescence emission spectra were recorded with a spectrofluorometer MOS-450 from Biologic at room temperature from 570 to 690 nm upon excitation at either 402 nm for detection of protoporphyrin IX, or 416 nm for detection of Mg protoporphyrin IX and Mg protoporphyrin IX methylester or 440 nm for detection of chlorophyllide. HPLC analysis was as described except that elution was monitored by absorbance detection at 420 nm and by fluorescence detection (λexcitation 420 nm/λemission 595 nm or λexcitation 420 nm/λemission 625 nm). Standards of protorporphyrin IX, of Mg protoporphyrin IX, of Mg protoporphyrin IX methylester prepared and identified as described in (6) were used. When required, plants were incubated overnight with 10 mM ALA and 5 mM MgCl2 in 10 mM Hepes pH 7.0 before extraction.

[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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)