Indirect determination of magnetic susceptibility tensors in peroxidases: a novel approach to structure elucidation by NMR

 The chemical shifts of several ¹³C nuclei positioned α to the haems in oxidised cyanide complexes of horseradish peroxidase and lignin peroxidase are reported and analysed in terms of π molecular orbitals with perturbed D_4h symmetry. The additional contributions to the paramagnetic shifts of ¹³C nuclei in the vinyl groups which arise from conjugation with the porphyrin π molecular orbitals are discussed, and an empirical correction factor is derived from a number of other compounds which contain haems b. The orbital mixing parameter which is obtained from the analysis of the experimental ¹³C shifts is compared with the orientation of the axial histidine ligands in X-ray structures of related compounds and found to be close to the orientation of the normal to the histidine ring. Comparison with the magnetic axes determined by fitting the dipolar shifts of several protons which have been assigned previously also shows close agreement with the negative in-plane rotation of the magnetic y axis. It is therefore possible to obtain the approximate orientation of the magnetic axes from ¹³C resonances of the haem and hence to determine the dipolar shifts at any point in space with respect to the haem by using these axes together with the anisotropy of the magnetic susceptibility, which can be obtained by extrapolation from EPR g values. Excellent agreement is found between dipolar shifts obtained by fitting an empirical magnetic susceptibility tensor and predictions based on ¹³C NMR and EPR in the case of lignin peroxidase. The agreement is less good in the case of horseradish peroxidase, in which the empirical magnetic z axis appears to be tilted significantly away from the haem normal, though this may be due in part to the lack of accurate atomic coordinates. It is concluded that useful estimates of the magnetic susceptibility tensor may be obtained from ¹³C NMR and EPR studies even in large mammalian peroxidases for which no structural models are available. Received: 27 December 1995 / Accepted: 17 April 1996     

Reactions of the haloalcohols HO(CH2)nCl (n = 2, 3) with platinum(II) - alkynyl and -alkyne complexes. X-ray crystal structure of trans-[Pt(Me) {C(OCH2CH2Cl) (CH2Ph)} (PPh3)2] [BF4]

The chloro complexes trans-[Pt(Me)(Cl)(PPh₃)₂], after treatment with AgBF₄, react with 1-alkynes HC---C---R in the presence of NEt₃ to afford the corresponding acetylide derivatives trans-[Pt(Me) (C---C---R) (PPh₃)₂] (R = p-tolyl (1), Ph (2), C(CH₃)₃ (3)). These complexes, with the exception of the t-butylacetylide complex, react with the chloroalcohols HO(CH₂)_nCl (n = 2, 3) in the presence of 1 equiv. of HBF₄ to afford the alkyl(chloroalkoxy)carbene complexes trans-[Pt(Me) {C[O(CH₂)_nCl](CH₂R) } (PPh₃)₂][BF₄] (R = p-tolyl, N = 2 (4), N = 3 (5); R=Ph, N = 2 (6)). A similar reaction of the bis(acetylide) complex trans-[Pt(C---C---Ph)₂(PMe₂Ph)₂] with 2 equiv. HBF₄ and 3-chloro-1-propanol affords trans-[Pt(C---CPh) {C(OCH₂CH₂CH₂Cl)(CH₂Ph) } (PMe₂Ph)₂][BF₄] (7). T alkyl(chloroalkoxy)-carbene complex trans-[Pt(Me) {C(OCH₂CH₂Cl)(CH₂Ph) } (PPh₃)₂][BF₄] (8) is formed by reaction of trans-[Pt(Me)(Cl)(PPh₃)₂], after treatment with AgBF₄ in HOCH₂CH₂Cl, with phenylacetylene in the presence of 1 equiv. of n-BuLi. The reaction of the dimer [Pt(Cl)(μ-Cl)(PMe₂Ph)]₂ with p-tolylacetylene and 3-chloro-1-propanol yields cis-[PtCl₂{C(OCH₂CH₂CH₂Cl)(CH₂C₆H₄-p-Me}(PMe₂Ph)] (9). The X-ray molecular structure of (8) has been determined. It crystallizes in the orthorhombic system, space group Pna2₁, with a = 11.785(2), B = 29.418(4), C = 15.409(3) Å, V = 4889(1) ų and Z = 4. The carbene ligand is perpendicular to the Pt(II) coordination plane; the PtC(carbene) bond distance is 2.01(1) Å and the short C(carbene)-O bond distance of 1.30(1) Å suggests extensive electronic delocalization within the Pt---C(carbene)---O moietry.     

Hematology and plasma chemistry values in free-ranging Humboldt penguins (Spheniscus humboldti) in Chile

To establish baseline hematologic and plasma biochemistry values in free-ranging Humboldt penguins (Spheniscus humboldti), heparinized blood samples were collected from 51 apparently healthy, adult Humboldt penguins residing at two colonies off the Chilean coast. Thirty samples were collected in April, 1992, from penguins inhabiting the Ex-islote de los Pájaros Niños in Algarrobo, Chile. In September, 1992, 21 samples were collected from birds inhabiting Isla de Cachagua, Chile. Hematologic values measured include packed cell volume, leucocyte count, leucocyte differential, and the presence of blood parasites. Plasma biochemistry values measured include glucose, blood urea nitrogen, creatinine, uric acid, calcium, inorganic phosphorous, sodium, potassium, chloride, total protein, albumin, globulin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, and creatine kinase. Only the mean values for chloride and for the number of eosinophils differed significantly between the two sample groups. No blood parasites were seen. © 1995 Wiley-Liss, Inc.     

Fast atom bombardment combined with tandem mass spectrometry for the determination of nucleosides

The positive and negative ion fast atom bombardment (FAB) mass spectra and fast atom bombardment collisionally activated decomposition (CAD) spectra of a series of nucleosides and two dinucleotides are reported. The nucleosides studied are substituted forms of guanosine, adenosine, nebularine, tubercidin, uridine, and related pyrimidines. The FAB and CAD data both contain similar information. The CAD spectra are found to provide some structural information not found in the FAB mass spectra. Tandem mass spectrometry also allows emphasis to be put on weak fragments which are either not observed in the FAB mass spectrum or are lost in the matrix ion signals.     

X-inactivation patterns in lymphocytes and skin fibroblasts of three cases of X-autosome translocations with abnormal phenotypes

Summary X-inactivation patterns were studied by replication analyses both in lymphocytes and skin fibroblasts of two patients carrying balanced X-autosome translocations, t(X;10)-(pter;q11) and t(X;17)(q11;q11), and one patient with an unbalanced translocation t(X;22)(p21;q11). Preferential late replication of the normal X chromosome was found in lymphocytes of both patients carrying balanced translocations and in skin fibroblasts of the patient carrying the translocation t(X;17). However, skin fibroblasts of the patient with a translocation t(X;10) showed preferential late replication of the abnormal der(X) chromosome with no spreading of late replication to the autosomal segment. In the case of unbalanced translocation t(X;22) there was preferential late replication of the der(X) chromosome both in lymphocytes and skin fibroblasts. The abnormal phenotype of the patients is discussed in relation to the observed X-inactivation patterns and the variability of the patterns in different tissues.     

Freeze-preservation of rice cells: A physiological study of freeze-thawed cells

Rice ( Oryza sativa L.) cells returning to in vitro culture after preservation at superlow temperature in liquid nitrogen are characterized by a number of physiological alterations. These include: reduction in respiration and glucose uptake, loss of intracellular potassium, decrease in the cellular level of key metabolites (ATP, glucose-6-phosphate and pyruvate) and fragility of protoplasts following the action of cell wall-degrading enzymes.
Nevertheless, cell growth resumes after a short lag phase (2–4 days) with an actual 70–100% cell survival, thus indicating that the observed damage is not lethal and can be repaired in a short time.     

Characterization of purified guanine aminohydrolase.

Guanine aminohydrolase (GAH) (E.C. 3.5.4.3) was purified by affinity chromatography on 9-(p-β-aminoethoxyphenyl)guanine-Sepharose to a specific activity of 35.5 units/mg. The molecular weight of the enzyme was estimated to be 110,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) showed that the enzyme was composed of subunits with molecular weights of approximately 52,000. Data from SDS-gel electrophoresis in a discontinuous buffer system and from isoelectric focusing in the presence of 8-m urea indicated that more than one type of subunit were present. This was consistent with multiple forms of the native enzyme seen by electrophoresis and isoelectric focusing in polyacrylamide gels. The isoelectric points for the different forms of GAH were in the range of 4.65–4.85. Amino acid analyses showed cysteine to be the minimum amino acid and gave a calculated molecular weight for GAH of 53,016 when the assumption that there were four cysteines per subunit was made. Guanine, 8-azaguanine, and 6-thioguanine served as substrates for the enzyme but 3-deazaguanine, a potent competitive inhibitor of GAH, did not. Fluoride ion inhibited the enzyme in a noncompetitive manner, and this inhibition decreased as pH increased. Variation of the kinetic parameters with pH suggested that hydroxide ion might be the second substrate and that a functional group on the enzyme with a pK_a near 5.6 was involved in the reaction. The enzyme was inactivated by treatment with p-hydroxymercurobenzoate and by photooxidation in the presence of rose bengal. Two plausible mechanisms are proposed for the reaction catalyzed by GAH.     

Chemical modification of enzymes: Critical evaluation of the graphical correlation between residual enzyme activity and number of groups modified

In the study of chemical modification of enzymes and other biologically active proteins, plots of fractional residual activity as a function of number of groups modified per enzyme molecule are often used to establish a correlation between the chemical modification and enzyme inactivation reactions and to determine the stoichiometry of the modification reaction. This paper presents a critical examination of the underlying theoretical framework of such graphs. Whereas these plots are usually presented as linear functions, it is shown here that the general equation describing the relationship between inactivation and modification contains an exponential term; therefore, in the general case, the plot is actually a curve. It is suggested that caution be exercised in the interpretation of such plots and that equations such as those derived in the text be used to fit theoretical curves to the data, in order to maximize the information gained from chemical modification experiments.     

Somatic embryogenesis in suspension cultures of Gossypium klotzschianum anderss

Somatic embryoids differentiated in suspension cultures of G. klotzschianum after 3–4 weeks of culture in a liquid medium containing glutamine (optimally, 10–15 mM). Embryogenesis occurred after a preculture of callus on a medium containing 10 mg/l of the cytokinin, 2iP. The embryoids had meristematic regions, a well formed epidermis, and formed roots and vestigial leaves. Asparagine was much less effective than glutamine in promoting embryoid differentiation. The presence of 2,4-D in the medium resulted in increased vigor of the suspension cultures and subsequently in the formation of many embryoids, but does not seem to be necessary for somatic embryogenesis in cotton.Technical Article 14646 from the Texas Agricultural Experiment Station