POSSIBLE ORIGIN FOR PORPHIN DERIVATIVES IN PREBIOTIC CHEMISTRY – A COMPUTATIONAL STUDY

A set of chemical reactions is postulated to account for the formation of the macrocyclic porphin structure, basic to the pyrrole derivatives chlorophyll, protoporphyrin, heme and bilirubin, important in photosynthesis, respiration and digestion. A set of equations is given for the prebiotic synthesis of porphin derivatives from the simple molecules; cyanoacetylene, diacetylene, carbon monoxide and ammonia that have been detected in space. A number of isomers of hydrogenated porphin arise which may lose hydrogen to give ultimately porphin and its dehydrogenated derivative. The reactions, while not unique, provide a pathway which has been shown to be feasible from the overall enthalpy changes in the ZKE approximation at the HF and MP2/6-31G* level     

Opiate activity of [DAla2, delta zPhe4]-methionine-enkephalinamide.

When deoxygenated chloroform solution of bilirubin IXα (ZZ) was irradiated by blue light, ion-pair reversed-phase high pressure liquid chromatography technique revealed that the pigment was converted to a mixture containing IIIα, IXα, XIIIα (ZZ-configuration), and more polar geometric isomers (E-configuration). All azodipyrroles derived from each peak of Z- or E-configuration resulted in one of the exo- or endo-vinyl isomers, indicating that the bilirubin molecule is not affected by any of the phenomena except for geometric isomerization under this photochemical condition.     

Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX.

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN. Starting from a pure biliverdin IX dimethyl ester, the corresponding free acid of biliverdin IX or bilirubin IX was available within 3-4 h. Preparation of spectrally pure bile pigment required final TLC on acid-cleaned neutral TLC plates. The absorption spectra of the free acids and dimethyl esters of biliverdin IX in methanol showed a broad band at about 650 nm and a sharp band at about 375 nm. The long-wave-length band was extremely sensitive to the presence of strong acid. A 10-fold molar excess of HCl caused a 35- to 50-nm shift of the absorption maximum to longer wavelengths and near doubling of the maximum absorption. The molar absorption coefficients of biliverdins were identical for each free acid and dimethyl ester pair. In each case, Beer's law was followed in both methanol and acidified methanol. Methanol also proved to be a suitable solvent for spectroscopic determination of the non-alpha isomers of bilirubin IX. The wavelength of maximum absorption and molar absorption coefficient of each dipyrrolic ethyl anthranilate azo pigment derived from the various bilirubin IX isomers are also reported.     

Molecular flow resonance Raman effect from retinal and rhodopsin.

We have performed resonance enhanced Raman measurements of retinal isomers in solution (all-trans, 11-cis, 9-cis, and 13-cis) and cetyltrimethylammonium bromide (CTAB) detergent extracts of bovine rhodopsin near physiological temperatures (17 degrees C). In order to measure these photolabile systems, we have developed a general technique which allows Raman measurements of any photosensitive material. This technique involves imposing a molecular velocity transverse to the Raman exciting laser beam sufficient to ensure that any given molecule moves through the beam so that it has little probability of absorbing a photon. We have also measured the resonance Raman spectra of crystals of the same retinal isomers. The data show that each isomer has a distinct and characteristic Raman spectra and that the spectrum of 11-cis-retinal is quite similar but not identical with that of rhodopsin and similarly for 9-cis-retinal compared with isorhodopsin. In agreement with previous work, the Raman data demonstrate that retinal and opsin are joined by a protonated Schiff base. Due to the fact that the Raman spectra of 11-cis-retinal (solution) and rhodopsin show bands near 998 and 1018 cm(-1), a spectral region previously assigned to C-Me stretching motions, it is suggested that 11-cis-retinal in solution is compased of a mixture of 12-s-trans and 12-s-cis, and that the conformation of rhodopsin is (perhaps distorted) 12-s-trans.     

N-Alkylprotoporphyrin IX formation in 3,5-dicarbethoxy-1,4-dihydrocollidine-treated rats. Transfer of the alkyl group from the substrate to the porphyrin

Administration of 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) to rats causes the accumulation of N-methylprotoporphyrin IX, a potent inhibitor of ferrochelatase. To clarify the origin of the porphyrin N-methyl group, we have synthesized and administered to rats N-ethyl-3,5-dicarbethoxy-1,4-dihydrocollidine (N-ethyl DDC) and 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), the DDC analogue with a 4-ethyl rather than 4-methyl group. Only N-methylprotoporphyrin IX is isolated from rats treated with the former agent, and only N-ethylprotoporphyrin IX from those treated with the latter. All four isomers of N-ethylprotoporphyrin IX are formed biologically. The structure of the isolated porphyrins has been confirmed by complete spectroscopic comparison with the four synthetic isomers of N-ethylprotoporphyrin IX. DDEP has been shown to cause NADPH- and time-dependent in vitro loss of hepatic microsomal cytochrome P-450. These results unequivocally establish that the 4-alkyl groups in DDC and dDEP are the source of the N-alkyl group in N-methyl- and N-ethylprotoporphyrin IX, respectively, and strongly suggest that the alkyl group is transferred to the prosthetic heme of cytochrome P-450 during catalytic processing of the substrate by the enzyme. The mechanism of the group transfer is discussed.     

Resonance Raman spectroscopy of bilirubins: band assignments and application to bilirubin/lipid complexation

Resonance Raman spectra of bilirubins IX alpha, III alpha, and XIII alpha and mesobilirubin XIII alpha in alkaline aqueous and chloroform solutions are reported. Partial band assignments of bilirubin IX alpha are proposed. The model compounds confirm assignments of bands of the Raman spectrum of bilirubin IX alpha to each of the two different pyrromethenones. Resonance Raman spectra of mesobilirubin IV alpha, vinylneoxanthobilirubinic acid, and vinylisoneoxanthobilirubinic acid in alkaline aqueous solution and of the tetra-n-butylammonium salt of bilirubin IX alpha are used to define markers for the presence or absence of internal hydrogen bonds. Interaction of bilirubin dianion and sphingomyelin liposomes is studied. The Raman evidence suggests that in the bilirubin dianion/liposome complex the intramolecular hydrogen bonds between the propionate groups and the lactam NH/CO are ruptured. It is proposed that in the complex the bilirubin propionates form ion pairs with the quaternary ammonium ion of the choline moiety of sphingomyelin.     

The specificity of biliverdin reductase. The reduction of biliverdin XIII isomers

The substrate specificity of the different molecular forms of biliverdin reductase (bilirubin:NAD(P)+ oxidoreductase, EC 1.3.1.24) using biliverdin XIII alpha, XIII beta and XIII gamma was examined. It was found that molecular form 1 (the major form in normal rat liver) reduced biliverdin XIII alpha at a much higher rate than the other two isomers. Molecular form 2 (the minor form) reduced isomers XIII alpha and XIII beta at similar rates, while molecular form 3 (the major form induced by CoCl2 treatment) reduced the XIII beta isomer at a slightly higher rate than the XIII alpha isomer. Molecular forms 2 and 3, both reduced isomer XIII gamma more slowly than they reduced the XIII alpha and XIII beta isomers. These results are similar to those obtained previously using biliverdins IX alpha, IX beta and IX gamma, suggesting that biliverdin reductase specificity is related to the type of the isomer rather than to the series (IX or XIII) of the isomer.     

Urinary excretion of isomers of biliverdin after destruction in vivo of haemoproteins and haemin.

The amount and isomeric composition of urinary biliverdin in rabbits were analysed by h.p.l.c. Physiological values were maintained after the injection of haemin. On the other hand, when haemoglobins from several mammalian species were injected into rabbits, the excretion of biliverdin-IX alpha and biliverdin-IX beta were increased 6-18-fold and 32-66-fold respectively over physiological excretion. Injection of myoglobin resulted in a 44-fold increase in excretion of the IX alpha-isomer. Coupled oxidation with ascorbate of haemoglobin and myoglobin by oxygen produced mainly the IX alpha- and IX beta-isomers from haemoglobin and the IX alpha-isomer from myoglobin. The destruction of part of the haem from injected haemoproteins by non-enzymic chemical degradation would account for the observed respective increases in the excretion of biliverdin isomers. The excretion of biliverdin isomers after the injection of phenylhydrazine into rabbits was similar to that after the injection of haemoglobin.     

High-performance liquid chromatographic separation and quantification of the four biliverdin dimethyl ester isomers of the IX series

High-performance liquid chromatography (hplc) has been used to separate and quantificate the dimethyl ester (DME) derivatives of the four biliverdin isomers of the IX series: biliverdin-IXα, -IXβ, -IXγ, and -IXδ. Samples of 0.5 to 10.0 nmol of biliverdin DME were detected quantitatively upon elution by monitoring the absorbance at 375 nm. A technique was developed in which p-bromoacetanilide (Dupont's recommended test compound for their Zorbax column) is used as a marker for biliverdin-IXα DME. To facilitate quantification of biliverdin-IXβ DME, its extinction coefficient was determined. This method has been used to study biliverdin isomers in various biological species. High-resolution NMR (360 MHz) was used to further characterize the isomers.     

Differential inhibition of hepatic ferrochelatase by the isomers of N-ethylprotoporphyrin IX

The four isomers of N-ethylprotoporphyrin IX have been synthesized. The two isomers with the N-ethyl group on pyrrole rings A or B inhibit rat liver ferrochelatase as effectively as the corresponding N-methyl analogues, whereas those with the N-ethyl moiety on rings C or D are 30–100 times less effective. The ability of N-alkyl porphyrins to inhibit ferrochelatase thus depends not only on the size of the N-alkyl group but also on its precise location on the porphyrin face.     

Resonance Raman assignment and evidence for noncoupling of individual 2- and 4-vinyl vibrational modes in a monomeric cyanomethemoglobin

We have investigated the resonance Raman spectra of monomeric insect cyanomethemoglobins (CTT III and CTT IV) reconstituted with (1) protohemes IX selectively deuterated at the 4-vinyl as well as the 2,4-divinyls, (2) monovinyl-truncated hemes such as pemptoheme (2-hydrogen, 4-vinyl) and isopemptoheme (2-vinyl, 4-hydrogen), (3) symmetric hemes such as protoheme III (with 2- and 3-vinyls) and protoheme XIII (with 1- and 4-vinyls), and (4) hemes without 2- and 4-vinyls such as mesoheme IX, deuteroheme IX, 2,4-dimethyldeuteroheme IX, and 2,4-dibromodeuteroheme IX. Evidence is presented that the highly localized vinyl C = C stretching vibrations at the 2- and 4-positions of the heme in these cyanomet CTT hemoglobins are noncoupled and inequivalent; i.e., the 1631- and 1624-cm-1 lines have been assigned to 2-vinyl and 4-vinyl, respectively. The elimination of the 2-vinyl (in pemptoheme) or the 4-vinyl (in isopemptoheme) does not affect the C = C stretching frequency of the remaining vinyl. Furthermore, two low-frequency vinyl bending modes at 412 and 591 cm-1 exhibit greatly different resonance Raman intensities between 2-vinyl and 4-vinyl. The observed intensity at 412 cm-1 is primarily derived from 4-vinyl, whereas the 591-cm-1 line results exclusively from the 2-vinyl. Again, there is no significant coupling between 2-vinyl and 4-vinyl for these two bending modes.     

Significance of the endo-vinyl group of bilirubin in photochemical reactions.

In photochemical experiments on bilirubin III alpha (no endo-vinyl group), IX alpha (one endo-vinyl group) and XIII alpha (two endo-vinyl groups) and in the photochemical, thermal and catalytical reversion of their photoproducts under anaerobic conditions, much more instability and complexity of photoproducts of bilirubin XIII alpha were observed than for those of bilirubin IX alpha or III alpha. On the basis of present and previous results of photochemical experiments in vitro and the fact that large amounts of (EZ)-cyclobilirubin IX alpha appear in the bile during phototherapy of neonatal hyperbilirubinaemia [Onishi, Kawade, Itoh, Isobe & Sugiyama (1980) Biochem. J. 190, 527-532], it is concluded that the endo-vinyl group plays a crucial role in the photochemical reaction of bilirubin IX alpha. On reversed-phase high-pressure liquid chromatography of photoisomers, it was found that the retention times of geometric isomers and E-cyclized structural isomers were shortened compared with those of Z-isomer and E-isomer, respectively, as precursor substances.     

Identification of a group of tetracarboxylate porphyrins, containing one acetate and three propionate β-substituents, in faeces from patients with symptomatic cutaneous hepatic porphyria and from rats with porphyria due to hexachlorobenzene

1. Three tetracarboxylate porphyrins, apart from coproporphyrin, were isolated from the faeces of patients with symptomatic cutaneous hepatic porphyria and of rats with porphyria caused by hexachlorobenzene poisoning. The porphyrins were not present in the urine of the porphyric rats or in the faeces of control rats. 2. Two of the porphyrins were identified as tris-(2-carboxyethyl)-carboxymethyl ethyl trimethyl porphin (isocoproporphyrin) and tris-(2-carboxyethyl)-carboxymethyl trimethyl porphin (de-ethyliso-coproporphyrin). 3. The third porphyrin, previously reported to be an analogue of coproporphyrin in which one propionate group is replaced by an alpha- or beta-hydroxypropionate group, was shown to be tris-(2-carboxyethyl)-carboxymethyl-(1-hydroxyethyl)-trimethyl porphin (hydroxyisocoproporphyrin). 4. The order of beta-substituents around the porphin ring could not be determined for any of the compounds. 5. Evidence for the existence of a fourth porphyrin, tris-(2-carboxyethyl)-carboxymethyl trimethyl vinyl porphin (dehydroisocoproporphyrin), a postulated precursor of the others, is described. 6. It is suggested that dehydroisocoproporphyrinogen is produced by decarboxylation-dehydrogenation of one of the propionate groups of pentacarboxylate porphyrinogen III.     

Protoporphyrin ix as a possible ancient photosensitizer: Spectral and photochemical studies

Due to the potential special position of protoporphyrin IX in the evolution of photosynthesis, the absorption and fluorescence characteristics of this pigment and its complexes with human serum albumin (HSA) and basic proteinoid have been studied in parallel with their photochemical activity. The most significant change in the absorption spectrum of PP IX was the appearance of a new maximum at 455 (or 461) nm in the presence of HSA or proteinoid respectively. Some changes in the physicochemical properties of PP IX in different microenvironments have been detected by changes in fluorescence emission and excitation spectra (intensity, quantum yields, position of maxima). The increase of fluorescence quantum yield resulting from the formation of PP IX complexes with HSA or proteinoid correlates with the increase of their photochemical activity. Results obtained are discussed from the point of view of the early evolution of the photosynthetic apparatus.     

4-vinyl and 2,4-divinyl deuteration effects on the low frequency resonance Raman bands of myoglobin: correlation with the structure of vinyl group

Resonance Raman spectra of myoglobin (Mb) reconstituted with 4-vinyl and 2,4-divinyl deuterated protoheme IX were studied in several oxidation, spin, and ligation states (deoxyMb, MbO2, MbCO, metMbH2O, and metMbCN-) with special attention to the low frequency vibrations. Frequency shifts observed on deuteration enabled us to assign some Raman bands to vibrations specifically involving the 2- or 4-vinyl group. The most significant deuteration effect was found for a band around 410 cm-1 in the ferrous state, which loses intensity on 4-vinyl deuteration and is ascribed to a porphyrin in-plane vibration strongly coupled with the skeletal bend of the vinyl group at position 4. Such strong coupling implies that the vinyl group lies in the same plane as the pyrrole II ring, as in the crystalline state. Thus, frequency shifts on vinyl deuteration may be useful as a probe of the orientation of the vinyl group. Resonance Raman spectra of Mb coordinated with various sizes of isocyanides are interpreted in terms of vinyl orientational changes induced by ligation.     

Variable composition of heme oxygenases with different regiospecificities in Pseudomonas species

Heme oxygenases (HO) degrade heme yielding iron, carbon monoxide and one of four possible biliverdin (BV) isomers. Pseudomonas aeruginosa PAO1 is thus far the only organism to contain two HOs with different regiospecificities: BphO and PigA. While BphO cleaves heme to exclusively yield BV IXα, PigA produces the BV isomers IXβ and IXδ. We bioinformatically identified putative HOs in diverse Pseudomonas strains, tested their enzymatic functionality and determined their regiospecificity. Surprisingly, even high amino acid sequence identities to the P. aeruginosa HOs were not sufficient to correctly predict the HO regiospecificity in all cases. Based on our results, Pseudomonas strains differ in their HO composition containing either BphO or PigA or both HO types. Concomitantly with the existence of bphO is the occurrence of at least one gene encoding a bacterial phytochrome implying that only BV IXα is the sufficient phytochrome chromophore. In contrast, pigA genes are organized in gene clusters associated with iron utilization implying a role of PigA in iron acquisition. However, at least in strains containing no PigA this function maybe fulfilled by BphO. Only a combination of homology searches and analyses of genetic environments is appropriate for a reliable prediction of the regiospecificity of Pseudomonas HOs.     

Fatty acids,Part 22: Infrared and Raman studies of all positional isomers of furan-containing C18 fatty esters

The infrared and Raman spectra of all 2,5-disubstituted C₁₈ furanoid fatty esters were studied. Where the furan system was located at the end of the alkyl chain or conjugated to the carbomethoxy group, such positional isomers could be readily identified through their unique absorption features in the infrared and Raman spectra. Other positional isomers of the series gave less differentiating spectral absorption characteristics.     

The gene for polypeptide IX of human adenovirus type 7

This paper describes the nucleotide sequence of subgroup B human adenovirus type 7 (Ad7) between positions 3351-4010, and of cloned cDNA derived from mRNAs encoded by this segment. One of these (mRNA VII) is shown to be unspliced, and has its 5'- and 3'-ends encoded by positions 3460 (determined by the nuclease S1 technique) and 3939, respectively. The mRNA sequence contains a single open reading frame for protein biosynthesis between the first available initiation triplet AUG at position 3481 to the stop codon UAA at position 3895. It can specify a polypeptide of 138 amino acids (14 098 daltons) which must be polypeptide IX of Ad7, as is evident from its mapping position, and from a comparison with the sequence of the protein IX gene of subgroup C adenovirus type 5.     

erythro-β-hydroxyaspartic acid in bovine factor IX and factor X

To localize β-hydroxyaspartic acid in factor IX and factor X the two proteins were cleaved with cyanogen bromide and trypsin, respectively. Peptides containing β-hydroxyaspartic acid were isolated and subjected to Edman degradation. The phenylthiohydantoin derivative of β-hydroxyaspartic acid was identified by HPLC in position 3 in the factor IX fragment and in position 1 in the factor X fragment. This corresponds to position 64 in factor IX and position 63 in the light chain of factor X. The assignments were confirmed by subtractive Edman degradation and with the dansyl method.     

Resonance Raman studies of ETE dehydrogenase (an iron sulfur flavoprotein)

ETF Dehydrogenase is an iron sulfur flavoprotein responsible for the transfer of electrons between electron transfer flavoprotein (ETF) and CoQ of the electron transport chain. We have determined the resonance Raman spectrum of this enzyme observing in the process at least seven of thirteen flavin bands in the 1100cm-1-1600 cm-1 region of the Raman spectrum. The positions of three of these bands, II, IX, and X (see Figure I and Table I for band numbering system) in ETF dehydrogenase is very similar to their positions in aqueous solution of flavins in which water is hydrogen bonded to N-1, N-5, C=0(2), C=0(4), and N-H(3) of flavin. Conversely the positions of the flavin Raman bands are considerably shifted from those of flavin in nonhydrogen bonding solvent. The positions of bands II, IX, and X are nearly identical to those in the flavoprotein glutathione reductase; x-ray structural investigations on this enzyme indicate that there is extensive hydrogen bonding between FAD and protein in this molecule. A previous study in our laboratory has demonstrated that metal complexation at N-5 and C=0(4) with either Ru or Ag produces large shifts in the positions of Raman bands II, VI, IX, and X. None of these shifts are observed in ETF dehydrogenase indicating that there is no direct inner sphere coordination of Fe to flavin. In addition to the Raman bands of flavin observed in our spectrum, we also observe one band that is in the Fe-S stretching region observed for a variety of Fe-S proteins. This band is located at 331 cm-1. The frequency of the band corresponds to the 335 cm-1 band associated with the strongest Fe-S stretching mode in the 4Fe-4S protein ferrodoxin from C. pasterianum. The observed frequency is quite different from that of the 3Fe-3S proteins such as ferrodoxin(II) from D. gigas. Finally, ETF dehydrogenase shows no loss of activity or visual evidence of photodegradation in the laser beam as most other FeS proteins do.