Procedure 3 Purification of Recombinant ALAD from E coli

Bacterial growth E. coli strain TB1 containing the plasmid pUC19 harboring the E. coli hemB gene in a modified EcoRI-B mHI fragment (Table 1) as constructed by Li et al. (20) is grown in 500 mL of LB medium containing ampicillin (50 pg mL) for 24 hours after inoculation from a starter culture. 2. Harvesting and cell lysis The cells are harvested by centrifugation at 3000x g for 30 minutes and washed to remove excess medium. Approximately 4 g of cell paste are then suspended in 20 mL of 50 mM...

Type I and Type III Hexacarboxylic Acid Porphyrins

There are two type I and six type III hexacarboxylic acid porphyrin isomers. The two type I isomers (6Iab and 6Iac) have been separated from the most abundant type III isomer (6IIIad) by isocratic RP-HPLC with 16 (vol vol) acetonitrile in 1 M ammonium acetate buffer, pH 5.16, as eluent on a Hypersil-ODS column (ThermoQuest, Bellafonte, PA, USA). The complete separation of all 8 isomers has not been achieved. Using the above system, 6IIIac coeluted with 6IIIbd, and 6IIIab coeluted with 6IIIbc...

Chemical Syntheses Of Porphyrins

Porphyrin chemical synthesis will be discussed here in connection with two series of compounds (i) those porphyrins which have been most often used in connection with model studies, e.g., 5,10,15,20-tetra-phenylporphyrin (TPP) 51 and 2,3,7,8, 12,13,17,18-octaethylporphyrin (OEP) 52 and (ii) those related to protoporphyrin IX 1 . Simply based on the symmetry in the substituent arrays of 51 and 52 and the lack of symmetry in 1 , it is obvious that it would be a waste of time to approach the...

References

Reiss-Husson. 1974. Influence of iron deficiency on the cytochromes of Rhodopseudomonasspheroides. Eur. J. Biochem. 47 573580. 2.Aono, S., H. Nakajima, K. Saito, and M. Okada. 1996. A novel heme protein that acts as a carbon monoxide-dependent transcriptional activator in Rho-dospirillum rubrum. Biochem. Biophys. Res. Commun. 228 752-756. 3.Barnes, H.J., M.P. Arlotto, and M.R. Waterman. 1991. Expression and enzymatic activity of recombinant cytochrome P450 17...

Analysis Of Other Tetrapyrroles

The quantitative determination of the other intermediates of the Chl biosynthetic pathway (Figure 1) can be carried out using essentially the same procedure as described for Proto. Table 1 reports the absorption coefficients for the compounds in various solvents, while Table 2 depicts the equations used to deconvolute the spectra for quantitative determinations. For each compound, the analysis involves the following steps 1. Extraction in ammoniacal acetone from tissues or organelles. 2....

Analysis Of Pchlide Ester a

The protochlorophyllide ester (Pch-lide E) a pool is a highly heterogeneous fully esterified tetrapyrrole pool. It is present in all etiolated and green tissues so far surveyed (Rebeiz, unpublished). It consists of MV and DV Pchlide a E (Figure 4) (10). The long chain fatty alcohols esterified at position 7 of the macrocycle are variable and have been reported to consist of ger-anylgeraniol (GG), dihydroGG (DHGG), tetrahydroGG (THGG), and hexahy-droGG (i.e., phytol) in Scenedesmus obliqu-us...

Info

Of the observation that MBV IXa, in contrast to BV IXa, is not further metabolized in these preparations. A typical reaction buffer contains 10 pM mesohemin IX in 20 mM Tris-HCl, pH 7.6, with 5 mM L-ascorbate, 0.1 mg mL catalase, 0.5 mg mL BSA, 10 (wt vol) glycerol, and reducing power provided by an NADPH-regenerat-ing system (10 mM glucose 6-phosphate, 0.5 mM NADP+, and 0.3 U mL glucose-6-phosphate dehydrogenase), together with 5 pg mL Porphyra umbilicalis ferredoxin, and 0.01 U mL spinach...

Procedure 5 Purification of His Tagged UROS from E coli

Bacterial growth The bacteria are grown from a starter culture in 2-L baffled flasks containing 1 L of LB media (with appropriate antibiotics) at 37 C with vigorous shaking until an A600 0.6 is reached, at which point isopropyl-P-D-thiogalactoside (IPTG) is added to a final concentration of 0.4 mM, and the cells are grown for another 2 hours. 2. Harvesting and cell lysis The bacteria are collected by centrifugation (10 000x g at 4 C). The bacterial pellet is resus-pended in 10 mL of binding...

Type I and Type III Pentacarboxylic Acid Porphyrins

There are four type III and one type I pentacarboxylic acid porphyrin isomers. These 5 isomers have been separated by RP-HPLC on a Hypersil-ODS column with 45 (vol vol) acetonitrile methanol (10 90) in 1 M ammonium acetate buffer, pH 5.16, as eluent (Figure 3 a). The elution order was 5I, 5bcd, 5abc, 5acd, and 5abd. A reversal of elution order between 5I and 5abd was observed when 19 acetonitrile in 1 M ammonium acetate, pH 5.16, was used as the mobile phase (Figure 3b). The presence of...

Growth Of 2d Crystals

Molecular Packing

2D crystals can be defined as crystals in which a motif, a protein, or assembly of proteins in the case of a protein crystal, is repeated at the points of a 2D lattice. Thus, a 2D crystal is one that is characterized by a unit cell that periodically repeats at precise locations in two dimensions but not in the third. Note that a 2D crystal is not a 2D object but is 3D. The nomenclature used to describe space groups of 2D crystals was first described by Holser (45) and revived by Fuller et al....

Protoporphyrin Heme and Related Compounds

Dicarboxylic acid porphyrins, heme, and related compounds are much more hydro-phobic than the other porphyrins described above. They require a much higher proportion of organic modifier for elution. Since acetonitrile is immiscible with 1 M ammonium acetate above the proportion of about 35 , it cannot be used as the sole organic modifier for the separation of this group of compounds. Either a mixture of acetoni-trile methanol (10 90) or methanol alone can be used instead. Methanol is completely...

Purification of CPO

Although the human CPO is thought to be associated with the outer surface of the inner membrane of the mitochondrion, when expressed in E. coli it is easily solubi-lized in the presence of 0.5 n-octyl-P-D-glucopyranoside (22). Recombinant expression of the human CPO was achieved by cloning the cDNA into the expression vector pBTac such that the cDNA was cloned in-frame with a 6-histi-dine tag at the 5' end (Table 1). The resulting plasmid, termed pHHCPO, was transformed into E. coli JM109....

Assay of CPO and Synthesis of Protoporphyrinogen

The incubation for the synthesis of pro-toporphyrinogen is undertaken in 50 mM Tris-HCl, pH 8.0, containing 0.2 Tween 20 and 2.5 mM glutathione. Coproporphyrinogen III is generated by the reduction of coproporphyrin III dihy-drochloride (Porphyrin Products, Logan, UT, USA) with 3 sodium amalgam. The reduction itself should be undertaken in 100 Tris-HCl, pH 8.0, and once the solution turns colorless, or nearly colorless, the solution is passed through a small 10-mL column of glass wool. This not...

HPLC Analysis and Purification

Bilins are readily separated by C18 reverse phase HPLC using isocratic solvent systems, and their distinctive absorption spectra make them easy to detect in the visible wavelength range. The most commonly used solvent system for analyzing most bilins is that of Beale and Cornejo (8), who separated phycobilins on an Altex Ultras-phere octadecylsilane (ODS) column (250 mm long x 46 mm diameter, 5 pm particle size). Separation was achieved using a mobile phase of acetone ethanol water acetic acid...

Analysis Of Pheophytins And Pheophorbides a

The first degradation products of Chl a are pheophytin a and or pheophorbides a Pheo(bides) a (40). Pheophytins are demetalated Chls while pheophorbides are demetalated Chlides. During senescence or after treatment with certain chemicals, green plants tend to accumulate substantial amounts of pheophytins and pheophor-bides (3). Spectrofluorometric techniques have been developed to determine the concentration of pheophorbides and pheo-phytins in crude extracts containing Chlides and Chls....

Aminolevulinic Acid And Porphobilinogen

ALA and porphobilinogen (PBG) are usually separated by ion exchange chro- Heme, Chlorophyll, and Bilins Methods and Protocols Edited by A.G. Smith and M. Witty 2002 Humana Press, Totowa, NJ matography, converted into the p-dimeth-ylamino-benzaldehyde derivatives, and then determined spectrophotometrically at 553 nm (16). The procedures, widely described in textbooks, are also available, with technical instructions, from ion exchange resins suppliers, e.g., Bio-Rad Laboratories (Hercules, CA,...

Anionic Compounds from Natural Porphyrins

Anionic porphyrins, metalloporphyrins, and their derivatives from natural sources have found a wide variety of usage in modern medicine and biochemistry including the field of photodynamic therapy for various disease states, heme oxygenase inhibition for prevention of jaundice, and inhibition and induction of this enzyme as a tool for biochemical research. Some metal-loporphyrins have been used as dioxygen detectors in fluids or air via phosphorescence quenching and as MRI contrast agents (47)....

Tetrapyrrole Structure And Function

Tetrapyrroles are a group of organic molecules that includes chlorophyll (Figure 1), hemes (Figure 2), bilins (Figure 3), and corrins, such as vitamin B12 (37). These molecules are also often referred to as por-phyrins, although strictly, these are only those compounds with the same oxidation state as heme. Chlorophyll, for example, has one more saturated bond and is therefore a chlorin (30). A pyrrole is a 5-membered ring containing one nitrogen, which is colorless, but when four pyrroles are...

Retention Mechanism Of Porphyrins And Metalloporphyrins In Rphplc

Understanding the retention behavior and mechanism is useful in the prediction and elucidation of the possible nature of substituent groups present in unknown porphyrins. The most dominant mechanism of retention in RP-HPLC is hydrophobic interaction. In porphyrins, this is between the side-chain substituents and the hydrophobic hydrocarbonaceous (ODS) stationary phase surface. The hydrophobic-ity of the porphyrin side-chain substituents increases in the order The number of the most hydrophobic...

Water Insoluble Porphyrins

Adler's DMF method is often employed for insertion of various metal ions into water-insoluble porphyrins (4). The free base porphyrin and a metal salt (acetate, chloride) are refluxed in DMF until the absorption spectra indicates that metala-tion is complete. The addition of water to the cooled solution precipitates the metal-loporphyrin, which can then be purified by chromatography. An example of this procedure is given below. One or two molecules of dimethylamine are often found bound to...

Behavior in Solution at the Molecular Level

Under certain conditions, porphyrins and metalloporphyrins undergo intermolecular association in solution. In water at pH 7.5 in 0.01 M Tris buffer, plots of absorbance versus concentration for H2-TPPS3 follow Beers law from approximately 5 x 10-7 M to 1.4 x 10-5 M, and the compound is considered monomeric (90). In the presence of 0.1 M KNO3 at this pH, however, increasingly negative deviations from Beers law are observed as the concentration of the porphyrin increases, consistent with a...

Analysis Of Porphyrins And Metalloporphyrins

Porphyrin Mass Spectrometry

It is often important to know the concentration in solution of a particular porphyrin or metalloporphyrin. Since this class of molecules often contains various kinds and amounts of nonporphyrin material that are isolated along with the porphyrin itself, simply weighing out a given amount of compound may give inaccurate results. With water-soluble porphyrins, water molecules are usually indicated by chemical analysis of the isolated solids. The anionic water-soluble H2-TPPS4 (Figure 1) analyzes...

Michael Witty

Department of Biochemistry, University of Cambridge, Cambridge, UK Humana Press Totowa, New Jersey 2002 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences arising from the information or opinions...

Purification of ALAD from E coli

Traditionally, ALADs have been isolated from sources that make large quantities of either heme or chlorophyll, such as liver, erythrocytes, and plants. However, more recent cloning strategies have led to the production of large quantities of recombinant forms of the enzyme. In this section, we will detail the purification of a recombinant version of the E. coli ALAD. Because of the way the protein is folded, it is not possible to tag the enzyme, for instance, with a polyhistidine epitope to...

K4

The equilibria are measured spectrophoto-metrically, and it is important to make sure that the porphyrin is monomeric under the pH titration conditions, and that the buffers used do not complex with the por-phyrins (56). For example, H2-TMPyP(4) shows pK4 0.8, pK3 1.4, while for the more basic H2-TAPP4+, pK4 3.95 and pK3 4.11. The pK2 and pK1 values for most porphyrins are above 12, although the H2- P-Br8-TMPyP(4) 4+ with eight electron-withdrawing bromines on the P-pyrrole positions (96) gives...

Chromatographic Separation of Pchlide a E from other MgPorphyrins

The mixed DV-MV Pchlide a E pool can be separated from other fully esterified Mg-porphyrins on thin layers of silica gel H as described in section 5.3.1 (10). The Pchlide a E, Chl a and b, and Pchlide a pools move with respective Rfs of about 0.9, 0.8, and 0.5. Pchlide a E is eluted with diethyl ether. After separation on thin layers of silica gel H, the Pchlide a E can be purified further on polyethylene plates. The ether extract is washed with 0.5 M KH2PO4 buffer, pH 7.0, to remove traces of...

Procedure 6 Pretreatment of the Dialysis Membranes

Heat the dialysis tubing (molecular weight cutoff 6000-8000, Spectra Por 1) in a 2-L beaker of boiling 50 ethanol for 1 hour. Use a 1-L beaker containing water to weigh down the dialysis tubing. 2. Rinse the dialysis tubing well with several changes of distilled water. 3. Heat the dialysis tubing in boiling 10 mM Na2CO3, 1 mM EDTA for 1 hour. 4. Rinse with distilled water as before. 5. Heat the dialysis tubing in boiling distilled water for 1 hour. 6. Store in distilled water containing 0.05...

Chromatography

Most porphyrins are purified by some form of chromatography. Column chro-matography is used for isolation of 100 mg to multigram quantities of material. Thin layer chromatography (TLC) on coated glass plates is employed in the 1 to 100 mg range and is valuable for the separation of compounds having similar retention factor (Rf) values. High-pressure liquid chromatography (HPLC) can separate and quanti-tate samples on the microgram scale. Many porphyrins and diamagnetic metallopor-phyrins...

And Anionic Porphyrins

From health considerations, we mention again that one should always wear inexpensive disposable gloves when working with porphyrins and the reagents and solvents involved in porphyrin synthesis. Most work should be done in a well-ventilated hood. The H2-TMPyP(X)4+ compounds are made from the w w-tetrakis(4-pyridyl)por-phyrin, H2-TPyP(4), and its isomers. These are prepared by refluxing, for 45 minutes, a propionic acid solution which is 0.24 M in pyrrole and 0.24 M in the 4(3 or...

Materials

A variety of polymers have been used to prepare 2-phase systems (see Table 2). PEG, a linear synthetic polymer of ethyl-ene oxide units, and Dx, poly(a-1,6-glu-cose), are the most commonly used polymers in the preparation of aqueous 2-phase systems. Examples of other sugar polymers used are Ficoll (polysucrose), pullulan, and maltodextrins. Derivatized carbohydrate polymers have also been used these include methylcellulose, hydroxyethylcel-lulose (HEC), Reppal PES (hydroxypropyl starch),...

Enzyme Assay and Incubation Protocol

ALA may be generated, using ALAS, with the following incubation mixture and substrates. 1. Incubation mixture Stock reaction buffer (100 pL) consisting of 20 mM potassium phosphate buffer, pH 7.2, containing 0.5 mM pyridoxal 5' phosphate, and 250 mM glycine is mixed with 5 pL of purified ALAS, and the reaction is initiated by the addition of 25 pL of 10 mM succinyl-CoA. Incubation is carried out at 37 C for up to 30 minutes. The incubation can be scaled up as required for the synthesis of ALA....

Introduction

There are thousands of porphyrins and metalloporphyrins, and hundreds of new derivatives appear each year. This variety arises because the cyclic conjugated tetra-pyrrole nucleus (Figure 1) can have different substituents at the eight P-pyrrole positions, at the four meso 5,10,15,20 carbon atoms, and N-alkyl groups can be added to the four central nitrogen atoms. Since its synthesis in 1972, over 8000 literature references have appeared on 5,10,15, compounds, and a similar number on its...

Procedure 1 Preparation of E coli Lysate Containing Recombinant R spheroides ALAS

Bacterial growth From an agar plate of recombinant E. coli harboring the R. spheroides hemA gene (JMA18), a single bacterial colony is removed and used to inoculate a starter culture (5 mL) of Luria-Bertani (LB) medium containing 50 pg mL ampicillin. 2. The culture is grown for between 5 to 10 hours at 37 C and then used to inoculate a larger (1 L) culture, which is grown overnight at 37 C with rotary shaking (160-180 rpm) for 18 hours. 3. Harvesting and cell lysis The cells are harvested by...

Procedure 3 Renaturing Lyophilized Phycocyanin Subunits

Dissolve lyophilized PC in 5 mM Na-phosphate, 1 mM P-mercaptoethanol, pH 7.0, and allow to stand overnight at 4 C. 2. Remove insoluble material by centrifu-gation. 3. Loaded the protein solution onto a DEAE cellulose DE-52 column (0.5 x 5 cm Whatman, Clifton, NJ, USA) equilibrated in 5 mM Na-phosphate, 1 mM P-mercaptoethanol, pH 7.0. 4. Subunits can be eluted immediately with 200 mM Na-phosphate, 1 mM -mercaptoethanol, pH 7.0. A large proportion of blue nonfluorescent material is typically...

Zinc Blot Assay for Bilin Attachment

Early examination of bile pigments revealed that bilatrienes form intensely fluorescent complexes with zinc ions in the presence of iodine (1). This and related observations have led to the examination of fluorescent zinc complexes of bilin-linked polypeptides. The initial work was performed using standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels with the fluorescent products being observed following UV illumination (2). The purpose of SDS-PAGE is to remove the...

Separation of Porphyrin Mixtures from Uroporphyrin to Protoporphyrin

From uroporphyrin to protoporphyrin, the compounds differ widely in hydropho-bicity. Gradient elution is therefore essential for the simultaneous separation of these porphyrins, including their isomers. A 15-minute linear gradient elution from 13 to 30 acetonitrile in 1 M ammonium acetate, pH 5.16, has been described for the separation of type I and type III isomers of 8-, 7-, 6-, 5-, and 4-car-boxylated porphyrins (7). The system is applicable to analysis where the separation of dicarboxylated...

Isolation And Analysis Of Chlorophyllprotein Complexes

As pointed out in the introduction to this chapter, there is a tremendous number of different isolation procedures described in the literature that have been devised for various Chl-containing complexes. It is impossible to give a complete overview of these techniques. Therefore, we will point to procedures that we believe are good examples of different separation principles used for Chl-proteins and focus on those techniques that are also useful for isolating reconstituted pigment protein...

Procedure 5 Pyridine Hemochrome Method

The isolated hemoprotein in a 4.0-mL sample volume is converted to a pyri-dine hemochrome by the addition of 0.5 mL of pyridine and 0.5 mL 0.5 N NaOH solution. 2. The reduced spectrum dithionite minus the oxidized spectrum no dithionite is calculated at 550 proto-heme or 557 nm heme c . The millimolar extinction coefficients Aeox-red at each of the wavelengths are 30.0 and 19.1 at 557 and 550 nm, respectively 87 . The method has also been applied to more complicated samples such as...

Procedure 7 TMBZ Staining of Polyacrylamide Gels

A solution of 6.3 mM TMBZ in methanol is prepared freshly prior to use. Immediately before use, 3 volumes of the TMBZ methanol solution is mixed with 7 parts of 0.25 M sodium acetate, pH 5.0. The gels are then immersed in this solution for a period of 1 to 2 hours in the dark and mixed every 15 minutes. 2. Following incubation, the gels are stained with the addition of H2O2 to a final concentration of 30 mM. The staining should be visible within 3 to 5 minutes with increasing intensity over...

Pigment Protein Complexes

The first pigment protein complexes which were reconstituted are the major LHCII of higher plants and the core LHC LH1 of photosynthetic bacteria. In these initial experiments, authentic proteins isolated from LH1 64 and total thylakoid membrane proteins 72 were employed. New possibilities were opened up by the availability of cDNAs for several LHC-apoproteins of different origins, which allowed the use of bacterially overexpressed LHC proteins in reconstitution experiments 17,68 . Since...

Preparation Of Porphyrins And Chlorins By Degradation Of Natural Pigments

It is truly fortunate that massive amounts of natural products containing both hemin 11 and chlorophylls a and b 12,13 can be accessed. Fischer's three volumes 20,22,24 , Die Chemie des Pyrrols, report an astonishing array of procedures for obtaining tetrapyrrole compounds from natural sources. Thus, large volumes of blood can be processed to provide hemin in kilogram quantities. From hemin, a large number of porphyrins and derivatives can be obtained see later . Similarly, chlorophyll...

Nalkylations To Prepare Cationic Porphyrins

It should be noted that all alkylating agents are hazardous, and extreme caution should be taken when working with these substances. Many workers methylate the H2-TPyP X compounds in hot or reflux-ing chloroform in the presence of excess CH3I, and the solid iodide salts of H2-TMPyP X precipitate from solution. Since the iodides are not very soluble in water, the product is stirred with the chloride form of an ion-exchange resin either in water, or in water-methanol, and warmed until the solid...

Phycobiliprotein Purification

Phycobiliproteins may exist in different aggregation states depending upon the individual type of biliprotein, the organism from which it was isolated, the composition of the solution containing it pH, ionic strength , and such factors as temperature and protein concentration. The purification of individual phycobiliproteins has been summarized previously 27 . A few minor improvements have been introduced using fast protein, peptide, and polynucleotide liquid chromatography FPLC Mono Q Amersham...

Hemoprotein Expression Systems 211 Oxygen Binding Proteins

The successful expression and purification of recombinant myoglobins from a number of species have been reported 103,110,122,127 . Initial studies on the expression in Escherichia coli of human myoglobin utilized a fusion protein consisting of the first 31 amino acids of the phage lambda cII gene and the tetrapeptide Ile-Glu-Gly-Arg, followed by the myoglo-bin gene sequence 122 . The fusion product was then isolated, reconstituted with heme, cleaved with trypsin, and purified to generate the...

Hemoproteins by Chemiluminescence

The detection of chemiluminescence is based on the peroxidase-dependent breakdown of luminol, and the reactions were carried out by modification of a standard procedure from the ECL blotting system commercially available from Amersham Pharmacia Biotech. 2. Gel electrophoresis SDS-PAGE gels, run with or without reducing agents, electroblots, or dotblots, are washed in Dulbecco's phosphate-buffered saline dPBS and incubated for 1 minute in a 1 1 mixture of CL reagents 1 and 2 ECL Blotting Kit...