K.G. Paul

The substrate profiles of the acidic and slightly basic horseradish peroxidases

Abstract 1. 1. The catalytic properties of two horseradish peroxidases, isoenzymes A2 and C2, with p I 3.9 and 8.8, have been compared. 2. 2. The rate of formation of Compound I has been determined for the hydrogen, methyl, ethyl, n -propyl, and hydroxymethyl hydroperoxides and for p -nitroperoxybenzoic acid. The elongation of the alkyl group hampered the reaction with the acidic peroxidase and stimulated the reaction with the basic peroxidase. The acidic peroxidase generally reacted more slowly. Its reaction with H 2 O 2 showed a higher energy of activation than that of the slightly basic peroxidase. 3. 3. The rate of reaction with hydrogen peroxide was slightly but definitely influenced by pH for both peroxidases. 4. 4. The rates of reaction of twelve hydrogen donors (phenols, aromatic amines, acidic substances) with the secondary peroxidase-peroxide complex of the two peroxidases could be arranged in three groups according to the chemical nature of the donors. The acidic peroxidase was consistently more active at pH 4.5 than at pH 7.0 whereas the slightly basic peroxidase showed the reverse behavior in the presence of phenols. 5. 5. It is concluded that the two peroxidases are kinetically different with distinct substrate profiles and that they may fulfill different physiological functions.

The isolation and some liganding properties of lactoperoxidase

Lactoperoxidase is an animal protein which participates in antimicrobial mechanisms [I]. Its electron donor profile differs from that of plant peroxidases regarding halide ions [2]. Isolation procedures are available but somewhat tedious [3--S]. Plant isoperoxidases with different affinities for aromatic substrates can be separated on phenylSepharoseR [6]. This observation has now been developed into an isolation procedure for LP. Its essential features are alternations between column materials with ionic and hydrophobic binding forces. The opposite requirements for ionic strength minimize the number of dialyses. The binding of LP to phenyland octyl-Sepharose is compared to the binding of horseradish peroxidase to both Sepharoses. Attempts are made to relate these adsorptions to optically operable equilibria between the peroxidases and free, aromatic ligands. Plant peroxidases can be isolated by means of affinity chromatography on hydroxamic acid-BioGel AR [7]. An imidazolecarrying polysaccharide binds hemoglobin and myoglobin specifically and unspecifically [8]. Sepharose-concanavalin A binds the glycoprotein HRP [9,10].

Optical, NMR and EPR properties of horseradish peroxidase and its donor complexes.

Complexes of horseradish peroxidase (HRP) and hydrogen donors have only recently been demonstrated spectrophotometrically [ 1,2]. The donor increased the dissociation of the HRl-cyanide complex and altered the optical spectrum of HRIHzOz compound II much more than that of free HRP, but no formation of an active ternary HRIHzOz -donor complex was assumed [ 11. EPR spectra of HRP without peroxide or donor (‘free’ enzyme) [2--63 have been variously interpreted as corresponding to high spin [2-41 or thermally mixed high and low spin species [6]. A transition from a multispecies EPR spectrum for the free enzyme to a pure high spin spectrum has been observed upon the addition of benzhydroxamic acid [2], but no EPR or NMR studies of HRI-donor complexes have been published. The present paper gives some optical, NMR, and EPR properties of HRP and its donor complex.** A recent analysis of the bacterial cytochrome c’ EPR spectrum is applied to the EPR spectrum of HRP [7,8].

Horseradish peroxidase with 2,4-modified haematins, including vinyl homologues

Abstract 1. 1. Some acyl and vinyl homologue porphyrins have been prepared. 2. 2. Pyridine is superior to alkali as a solvent for haemin to be used for the preparation of haemochrome. 3. 3. The Fe3+ and Fe2+ spectra of peroxidases with proto-, haemato-, meso-, deutero-, diacetyldeutero-, dipropenyldeutero- and dibutenyldeuterohaematins have been determined. Diacetyldeuterohaematin peroxidase gives a mixed-type spectrum which is slightly temperature dependent even above o°C. 4. 4. Meso-, proto- and diacetyldeuterohaematin peroxidases shift from the neutral to the alkaline form with p K 11.8, 11.1 and 9.0. 5. 5. At pH 4.5, k 1 (H2O2, stopped-flow) is essentially equal for the meso-, haemato-, deutero- and diacetyldeuterohaematin peroxidases and about twice as high for the three alkenyl peroxidases. At pH 7.0, k 1 of the first group remains unchanged, whereas k 1 of the second group increases, the first demonstration of a pH effect upon k 1 . 6. 6. k 4 (ascorbate) shows a 200-fold difference between the two extremes, dipropenyl- and diacetyldeuterohaematin peroxidase. The dihydroxyfumarate oxidase activity differs some 10 times between the extremes meso- and diacetyldeuterohaematin peroxidase, with only slight correlation to k 4 for ascorbate.

Umecyanin, a novel intensely blue copper protein from horseradish root

Purification and properties of umecyanin : a novel intensely blue copper protein from horseradish root

The vibrational bands of carbon monoxide bound to hemes or metal surfaces

Infrared spectra of imidazole carbonyl complexes of 2,4-substituted hemes are presented. An increased CO stretch frequency is accompanied by a lowered FeC vibrational energy. Hartree-Fock-Slater electron structure calculations discern pi and sigma contributions to the observed shifts of vibrational energies. We conclude that an enhanced electron availability manifests itself as a lowered ferric/ferrous reduction potential, increased filling of the 2 pi orbital of liganded CO which in turn reduces nu CO and increases nu Fec, and increased basicity of the liganded CO. Analogies between CO liganded to heme and CO adsorbed onto metal surfaces are discussed.

Mutant hemoglobin stability depends upon location and nature of single point mutation

The temperature dependence of the rates of heme release from the β subunits of methemoglobin A and 5 β mutant methemoglobins has been determined. The rates were largest fort wo hemoglobins with mutations distal to heme, previously known to be unstable. The other 3 mutants also released heme faster than A. These hemoglobins, with single point mutations at the α1β2 interface, were previously thought to be stable. The low reported yields of the 5 mutant protein covaries with the relative rates of heme release from the met species.

Spin and electron distributions in heme-cyanide models and hemeproteins

Proton NMR spectra of low-spin Fe(III) cyanoprotoheme as prosthetic group in a number of proteins are presented. The diagonally positioned 1-, 5- and 3-, 8-methyl groups obey shifts proportional to the Fe(III)/(II) reduction potential Em7, which indicates a pseudo-contact interaction. The correlation with Em7 is understandable if one postulates an enhanced rhombic distortion, dominating the Fe-methyl dipolar interactions. Hartree-Fock-Slater quantum chemical calculations show no significant changes of spin density as a function of the Fe-L5 distance, except at the iron atom and predominantly in the 3dxz and 3dyz orbitals. 4p orbitals, on the other hand, uphold most of the changes of electron density. We also observe a principal difference in the amino acid sequences in the heme-accommodating pocket of oxygen carriers and two-electron transmitters.

Fast heme release from inactive proteins

Kinetics for the release of the prosthetic group from hemoproteins is presented. Heme-protein separation is a biphasic reaction, where an initial phase is significantly faster than the dominant, slow phase. A previous communication concluded that the slow phase represents the active protein. This communication presents data for porphyrin release which shows that the initial fast phase represents an inactive form of the protein. Moreover, we suggest that the fast to slow phase ratio is a sensitive monitor of sample quality for many hemoproteins and that an extrapolation of the slow phase absorbance leads to new estimates for the true physical parameters of unperturbed proteins.

THE SPONTANEOUS HEMIN RELEASE FROM LUMBRICUS TERRESTRIS HEMOGLOBIN

The slow, spontaneous release of hemin from earthworm, Lumbricus terrestris, hemoglobin has been studied under mild conditions in the presence of excess apomyoglobin. This important protein is surprisingly unstable. The reaction is best described as hemin released from the globin into water, followed by quick engulfment by apomyoglobin. The energetics of this reaction are compared with those of other types of hemoglobins. Anomalously low activation energy and enthalpy are counterbalanced by a negative entropy. These values reflect significant low frequency protein motion and dynamics of earthworm hemoglobin and may also indicate an open structure distal to the heme. This is also supported by the infrared spectrum of the carbonyl hemoprotein, which indicates several types of distal interactions with the bound CO. The reported low heme to polypeptide ratio for this protein may be due to facile heme and hemin release by the circulating protein.