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 in nuclear-encoded photosynthetic gene expression [2]
Conversely, the level of LHCB mRNA was significantly reduced in the mutant due to transcriptional regulation. Since the mutant specifically accumulated Mgprotoporphyrin IX without any treatment, our results independently suggest that Mgprotoporphyrin IX is a negative regulator of nuclear photosynthetic gene expression, consistent with previous observations in plants treated with norfluzol. Furthermore, the chlm mutant behaved as a super-strong repressor of the LHCB promoter and appeared to suppress LHCB expression more effectively than plants treated with norfluzol. The difference in the degree of suppression may be due to the different levels of Mgprotoporphyrin IX accumulation in the plants. Another possibility is related to the complete absence of Mgprotoporphyrin IX methyl ester and its derivatives in the mutant. This suggests that one of these components is a positive regulator of nuclear photosynthetic gene expression. Mgprotoporphyrin methyl ester itself may be a positive effector. To support the latter hypothesis, we found that in the absence of norflurasone, the xanthan barley mutant (xanthan l) deficient in magnesium protoporphyrin methyl ester cyclase accumulated magnesium protoporphyrin IX methyl ester and had high levels of LHCB expression. In addition, we reported a positive correlation between LHCB expression and methyltransferase activity in tobacco CHLM antisense and sense RNA mutants. The activity of magnesium protoporphyrin IX methyltransferases obviously depends on the availability of magnesium protoporphyrin IX, but is also certainly regulated by the levels of Ado-Met and Ado-Hcy in chloroplasts (see, for example, reference 38). The different effects of Mg protoporphyrin IX and Mg protoporphyrin IX methyl ester on LHCB expression allow for fine regulation of one-carbon metabolism in the synthesis of photosynthetic complexes.

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Increased CHLH levels[2]
In the chlm mutant, we observed a significant increase in the level of mature CHLH, while the level of CHLH mRNA was slightly decreased. Our data suggest that stabilization of proteins in chloroplasts may have occurred in the chlm mutant. One possible explanation for this accumulation is the elevated level of Mgprotoporphyrin IX in the mutant. In fact, due to the undetectable presence of Mgprotoporphyrin intermediates under standard conditions, a tightly bound substrate channel between Mg chelase and methyltransferase has long been suspected. Recent studies have shown a physical interaction between CHLM and CHLH. During diurnal growth, Mg chelase activity peaks upon transition from darkness to light, while methyltransferase activity peaks several hours later. During this period, the level of Mgprotoporphyrin IX is transiently higher than that of Mgprotoporphyrin IX methyl ester. Mgprotoporphyrin IX may bind to CHLH, thereby preventing the transient accumulation of free Mgprotoporphyrin IX when chlorophyll single-chain (CHLM) content is insufficient/activity is low. This both stabilizes the rapidly turnover CHLH protein and protects it from severe photo-oxidative damage caused by free Mgprotoporphyrin IX. Regardless of the mechanism of CHLH accumulation, it is open to question whether its synchronous increase with magnesium protoporphyrin IX plays a role in the regulatory pathway mediating the transfer of plasmids to the nucleus. [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.)