Gunnel Sievers

The prosthetic group of milk lactoperoxidase is protoheme IX.

The prosthetic group of milk lactoperoxidase has been isolated from a Pronase hydrolysate of the enzyme and identified spectroscopically and chromatographically as protoheme IX. Partial degradation of the heme occurred during the proteolysis, possibly as a result of coupled oxidation in the presence of glycine and oxygen. The heme is assumed to be buried in the protein molecule in a crevice, which allows ligands to bind to the iron on one side only. The pyridine hemochrome of lactoperoxidase with alpha-band at 563 nm is interpreted as a mixed ligand complex with pyridine on one side of the heme and a ligand originating from the protein on the other. No experimental evidence supports the view that the heme is covalently bound to the protein through an ester linkage.

Structure of milk lactoperoxidase: Evidence for a single polypeptide chain

The structure of the compound responsible for the peroxidatic activity in milk has interested many laboratories ever since the first demonstration of the activity about a century ago. The enzyme,later called lactoperoxidase [ 11, is a hemoprotein and its prosthetic group has recently been established to be protoheme [2]. The structure of the protein moiety has also been investigated. The M,-value, obtained from sedimentation studies, iron content and amino acid composition, is approx. 78 000 [3,4], and it contains approx. 8-I 0% carbohydrates [3,4]. It has also been suggested that lactoperoxidase consists of two almost identical subunits and that the N-terminal amino acid of one chain is leucine and that of the other is blocked 131. The number of subunits with their rel. molecular masses can be determined in SDS-PAGE, and the MIvalue of lactoperoxidase as calculated by this method is reported here. The protein has also been cleaved with CNBr at the methionine residues and the&f*-value estimated from the resulting peptide fragments. The nature and the number of the N-terminal amino acids were determined by the dansylation method. The results indicate that lactoperoxidase consists of only one polypeptide chain with leucine at the amino end.

Structure of milk lactoperoxidase. A study using circular dichroism and difference absorption spectroscopy.

Circular dichroism spectra of milk lactoperoxidase, its fluoride and cyanide derivatives, and those of ferrous lactoperoxidase and its carbonyl and cyanide compounds, were recorded in the wavelength region 200-670 nm. All derivatives have split ellipticity bands, suggesting that lactoperoxidase has a narrow heme pocket that prevents ligands forming linear iron-ligand bonds. Difference absorption spectroscopy of the enzyme in the far-ultraviolet region supports the previously held view that the fifth ligand of the heme iron is histidine. The secondary structure of lactoperoxidase, calculated from the far-ultraviolet circular dichroism spectrum, contains 65% beta-structure, 23% alpha-helix and 12% unordered structure. Reduction of lactoperoxidase with dithionite gives two forms, indicating that after reduction some compound arising from dithionite binds in the vicinity of the heme iron.

Thin layer chromatography of free porphyrins

Abstract Thin layer chromatography procedures are described for the separation and identification of a number of porphyrin free acids. Dicarboxylic porphyrins have been separated on neutral plate in benzene−methanol−−formic acid and chloroform−methanol−formic a and on acidic plates in toluene−isopropanol, benzene−isopropanol and benzene−methanol. A straight−line relationship for the mobility of prophyrins containing from nought to four carboxyl groups in the prophyrin ring was obtained on neutral plates in benzene−methanol−formic acid.

Study of the pseudoperoxidatic activity of soybean leghemoglobin and sperm whale myoglobin.

The compound formation between soybean leghemoglobins a and c and H2O2 or ethyl hydroperoxide has been studied and compared with the hydrogen peroxide compound of sperm whale myoglobin. the titration data show that the hydrogen peroxide compounds of leghemoglobins are formed in a 1:1 molar ratio. The kinetics of the formation of the compounds follow first-order kinetics and the compounds are formed considerably faster than the myoglobin peroxide compound. The pseudoperoxidatic activity of leghemoglobins a and c and myoglobin was studied using guaiacol as electron donor. The maximal reaction velocities of leghemoglobins are greater than that of myoglobin. The results indicate that the peroxidatic activity of ferrileghemoglobin may be biologically important for instance in aging root nodules.

Resonance Raman study on yeast cytochrome c peroxidase Effect of coordination and axial ligands

Resonance Raman spectra are reported for native ferric cytochrome c peroxidase, its cyanide and fluoride compounds, those of the ferrous enzyme and its cyanide and carbonyl compounds, and the spectrum of the hydrogen peroxide compound, compound I. Band frequencies of ferric horseradish peroxidase isoenzyme C2 and its derivatives are also given. Comparison of the frequencies of the bands around 1400, 1500, 1560-1580, and 1610-1640 cm-1 with those of other hemoproteins and heme model compounds showed that in ferric highspin compounds in particular the bands are not only spin and oxidation sensitive, as has previously been reported, but that they also reflect the coordination of the heme iron. It is suggested that ferric cytochrome c peroxidase and horseradish peroxidase are pentacoordinated. In the hexacoordinated fluoride, cyanide and carbon monoxide derivatives the bands reflect the spin state and the out-of-plane position of the heme iron. The spectrum of cytochrome c peroxidase compound I supports previous studies that suggest that it has a lowspin heme iron in the Fe(IV) oxidation state.

Circular dichroism studies on cytochrome c peroxidase from baker's yeast (Saccharomyces cerevisiae)

Circular dichroism spectra of cytochrome c peroxidase from bakers yeast, those of the reduced enzyme, the carbonyl, cyanide and fluoride derivatives and the hydrogen peroxide compound, Compound I, have been recorded in the wavelength range 200 to 660 nm. All derivatives show negative Soret Cotton effects. The results suggest that the heme group is surrounded by tightly packed amino acid sidechains and that there is a histidine residue bound to the fifth coordination site of the heme iron. The native ferric enzyme is probably pentacoordinated. The circular dichroism spectra of the ligand compounds indicate that the ligands form a nonlinear bond to the heme iron as a result of steric hindrance in the vicinity of the heme. The spectrum of Compound I shows no perturbation of the porphyrin symmetry. The dichroic spectrum of the native enzyme in the far-ultraviolet wave-length region suggests that the secondary structure consists of roughly equal amounts of alpha-helical, beta-structure and unordered structure. After the removal of the heme group no great changes in the secondary structure can be observed.

Circular dichroism of soybean leghemoglobin

Circular dichroic (CD) spectra of soybean leghemoglobin, and some of its liganded derivatives were measured over the wavelength range of 650 to 200 nm. The heme-related circular dichroic bands in the visible, Soret and ultraviolet wavelength regions exhibit Cotton effects characteristic of each of the compounds examined. The positions of the dichroic bands vary with ligand substitutions and the oxidation state of the iron. All leghemoglobin derivatives, except the apoprotein, exhibit negative circular dichroic bands in the region of Soret absorption. In this region the optical activity of compounds with high-spin moments is greater than that of compounds with low or intermediate spin moments. The ellipticity of the heme band at about 260 nm is also altered by ligand binding and spin state. The dichroic spectra in the far-ultraviolet region indicated a high extent of alpha-helical structure (about 70%) in the native leghemoglobin and its liganded derivatives. The helicality of the apoprotein seems to diminish suggesting a decrease caused by the removal of the heme.

Optical and EPR spectroscopy studies on haem arginate, a new compound used for treatment of porphyria

A protohaem compound, used for treatment of porphyrias, has been studied to elucidate its state of aggregation. EPR and absorption spectroscopy measurements reveal that 38.3 mM protohaem, dissolved in 40% 1,2-propanediol/10% ethanol/water solution, also containing 153 mM arginine, is partly EPR silent. It exists as high molecular weight aggregates and probably also as mu-oxo-dimers. Dilution in the aqueous alcohol solution dissolves the aggregates first to oligomers and dimers, and finally to monomers (Kdiss = 24 X 10(-6)M). When haem is diluted in 0.9% sodium chloride, a fully monomeric state is not reached even at 1 microM concentration. At 3.5 microM concentration, that used for infusion in patients, the haem is still totally aggregated.

Conformations of Chlorophylls a and a′ and their Magnesium-Free Derivatives as Revealed by Circular Dichroism and Proton Magnetic Resonance

Abstract The electronic absorption (UV/VIS), circular dichroism (CD) and proton magnetic resonance (1H NMR) spectra have been recorded for C-10 epimeric chlorophylls a and a′, pheophytins a and a′ as well as pheophorbides a and a′. Although the epimers in each pair showed virtually identical UV/VIS spectra, their CD spectra were profoundly different and exhibited opposite signs at most wavelengths in the UV region. The differences were interpreted as arising, in part, from different C-10 configurations, and, in part, from conformational alterations induced by the steric strain in the crowded periphery of the macrocycle. The conformational alterations were also clearly indicated by the 1H NMR Δδ-values observed for the α,β and δ methine protons, the C-10 protons and most methyl group protons of the epimers in each pair. This was considered to imply changes in the geometry of the whole macrocycle. The Δδ-values were larger for the Mg-free epimers than for the chlorophyll epimers, which shows that the central Mg-atom makes the macrocycle more rigid. Correlations between the signs of the CD bands and configurations are discussed.