DNA-XPA interactions: a (31)P NMR and molecular modeling study of dCCAATAACC association with the minimal DNA-binding domain (M98-F219) of the nucleotide excision repair protein XPA.

Recent NMR-based, chemical shift mapping experiments with the minimal DNA-binding domain of XPA (XPA-MBD: M98-F219) suggest that a basic cleft located in the loop-rich subdomain plays a role in DNA-binding. Here, XPA-DNA interactions are further characterized by NMR spectroscopy from the vantage point of the DNA using a single-stranded DNA nonamer, dCCAATAACC (d9). Up to 2.5 molar equivalents of XPA-MBD was titrated into a solution of d9. A subset of (31)P resonances of d9 were observed to broaden and/or shift providing direct evidence that XPA-MBD binds d9 by a mechanism that perturbs the phosphodiester backbone of d9. The interior five residues of d9 broadened and/or shifted before (31)P resonances of phosphate groups at the termini, suggesting that when d9 is bound to XPA-MBD the internal residues assume a correlation time that is characteristic of the molecular weight of the complex while the residues at the termini undergo a fraying motion away from the surface of the protein on a timescale such that the line widths are more characteristic of the molecular weight of ssDNA. A molecular model of the XPA-MBD complex with d9 was calculated based on the (15)N (XPA-MBD) and (31)P (d9) chemical shift mapping studies and on the assumption that electrostatic interactions drive the complex formation. The model shows that a nine residue DNA oligomer fully covers the DNA-binding surface of XPA and that there may be an energetic advantage to binding DNA in the 3'-->5' direction rather than in the 5'-->3' direction (relative to XPA-MBD alpha-helix-3).

Human nucleotide excision repair protein XPA: NMR spectroscopic studies of an XPA fragment containing the ERCC1-binding region and the minimal DNA-binding domain (M59-F219).

XPA is a central protein component of nucleotide excision repair (NER), a ubiquitous, multi-component cellular pathway responsible for the removal and repair of many structurally distinct DNA lesions from the eukaryotic genome. The solution structure of the minimal DNA-binding domain of XPA (XPA-MBD: M98-F219) has recently been determined and chemical shift mapping experiments with 15N-labeled XPA-MBD show that XPA binds DNA along a basic surface located in the C-terminal loop-rich subdomain. Here, XPA-DNA interactions are further characterized using an XPA fragment containing the minimal DNA-binding domain plus the ERCC1-binding region (XPA-EM: M59-F219). The 15N/1H HSQC spectrum of XPA-EM closely maps onto the 15N/1H HSQC spectrum of XPA-MBD, suggesting the DNA-binding domain is intact in the larger XPA fragment. Such a conclusion is corroborated by chemical shift mapping experiments of XPA-EM with a single strand DNA oligomer, dCCAATAACC (d9), that show the same set of 15N/1H HSQC cross peaks are effected by the addition of DNA. However, relative to DNA-free XPA-MBD, the 15N/1H HSQC cross peaks of many of the basic residues in the loop-rich subdomain of DNA-free XPA-EM are less intense, or gone altogether, suggesting the acidic ERRC1-binding region of XPA-EM may associate transiently with the basic DNA-binding surface. While the DNA-binding domain in XPA-EM is structured and functional, 15N-edited NOESY spectra of XPA-EM indicate that the acidic ERRC1-binding region is unstructured. If the structural features observed for XPA-EM persist in XPA, transient intramolecular association of the ERCC1-binding domain with the DNA-binding region may play a role in the sequential assembly of the NER components.

Interactions of human nucleotide excision repair protein XPA with DNA and RPA70 Delta C327: chemical shift mapping and 15N NMR relaxation studies.

Human XPA is an essential component in the multienzyme nucleotide excision repair (NER) pathway. The solution structure of the minimal DNA binding domain of XPA (XPA-MBD: M98-F219) was recently determined [Buchko et al. (1998) Nucleic Acids Res. 26, 2779-2788, Ikegami et al. (1998) Nat. Struct. Biol. 5, 701-706] and shown to consist of a compact zinc-binding core and a loop-rich C-terminal subdomain connected by a linker sequence. Here, the solution structure of XPA-MBD was further refined using an entirely new class of restraints based on pseudocontact shifts measured in cobalt-substituted XPA-MBD. Using this structure, the surface of XPA-MBD which interacts with DNA and a fragment of the largest subunit of replication protein A (RPA70 Delta C327: M1-Y326) was determined using chemical shift mapping. DNA binding in XPA-MBD was highly localized in the loop-rich subdomain for DNA with or without a lesion [dihydrothymidine (dhT) or 6-4-thymidine-cytidine (64TC)], or with DNA in single- or double-stranded form, indicating that the character of the lesion itself is not the driving force for XPA binding DNA. RPA70 Delta C327 was found to contact regions in both the zinc-binding and loop-rich subdomains. Some overlap of the DNA and RPA70 Delta C327 binding regions was observed in the loop-rich subdomain, indicating a possible cooperative DNA-binding mode between XPA and RPA70 Delta C327. To complement the chemical shift mapping data, the backbone dynamics of free XPA-MBD and XPA-MBD bound to DNA oligomers containing dhT or 64TC lesions were investigated using 15N NMR relaxation data. The dynamic analyses for the XPA-MBD complexes with DNA revealed localized increases and decreases in S2 and an increase in the global correlation time. Regions of XPA-MBD with the largest increases in S2 overlapped regions having the largest chemical shifts changes upon binding DNA, indicating that the loop-rich subdomain becomes more rigid upon binding DNA. Interestingly, S2 decreased for some residues in the zinc-binding core upon DNA association, indicating a possible concerted structural rearrangement on binding DNA.

NMR studies of structure, hydrogen exchange, and main-chain dynamics in a disrupted-core mutant of thioredoxin.

Core-packing mutants of proteins often approach molten globule states, and hence may have attributes of folding intermediates. We have studied a core-packing mutant of thioredoxin, L78K, in which a leucine residue is substituted by lysine, using 15N heteronuclear two- and three-dimensional NMR. Chemical shift differences between the mutant and wild-type main-chain resonances reveal that structural changes caused by the mutation are localized within 12 A of the altered side chain. The majority of resonances are unchanged, as are many 1H-1H NOEs indicative of the main-chain fold, suggesting that the structure of L78K is largely similar to wild type. Hydrogen exchange studies reveal that residues comprising the central beta-sheet of both mutant and wild-type proteins constitute a local unfolding unit, but with the unfolding/folding equilibrium approximately 12 times larger in L78K. The dynamics of main-chain NH bonds in L78K were studied by 15N spin relaxation and compared with a previous study of wild type. Order parameters for angular motion of NH bonds in the mutant are on average lower than in wild type, suggesting greater spatial freedom on a rapid time scale, but may also be related to different rotational correlation times in the two proteins. There is also evidence of greater conformational exchange in the mutant. Differences between mutant and wild type in hydrogen exchange and main-chain dynamics are not confined to the vicinity of the mutation. We infer that mispacking of the protein core in one location affects local dynamics and stability throughout.

Characterizing the use of perdeuteration in NMR studies of large proteins: 13C, 15N and 1H assignments of human carbonic anhydrase II.

Perdeuteration of all non-exchangeable proton sites can significantly increase the size of proteins and protein complexes for which NMR resonance assignments and structural studies are possible. Backbone 1H, 15N, 13CO, 13C alpha and 13C beta chemical shifts and aliphatic side-chain 13C and 1H(N)/15N chemical shifts for human carbonic anhydrase II (HCA II), a 259 residue 29 kDa metalloenzyme, have been determined using a strategy based on 2D, 3D and 4D heteronuclear NMR experiments, and on perdeuterated 13C/15N-labeled protein. To date, HCA II is one of the largest monomeric proteins studied in detail by high-resolution NMR. Of the backbone resonances, 85% have been assigned using fully protonated 15N and 3C/15N-labeled protein in conjunction with established procedures based on now standard 2D and 3D NMR experiments. HCA II has been perdeuterated both to complete the backbone resonance assignment and to assign the aliphatic side-chain 13C and 1H(N)/15N resonances. The incorporation of 2H into HCA II dramatically decreases the rate of 13C and 1H(N)T2 relaxation. This, in turn, increases the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments. Many otherwise marginal heteronuclear 3D and 4D correlation experiments, which are important to the assignment strategy detailed herein, can now be executed successfully on HCA II. Further analysis suggests that, from the perspective of sensitivity, perdeuteration should allow other proteins with rotational correlation times significantly longer than HCA II (tau c = 11.4 ns) to be studied successfully with these experiments. Two different protocols have been used to characterize the secondary structure of HCA II from backbone chemical-shift data. Secondary structural elements determined in this manner compare favorably with those elements determined from a consensus analysis of the HCA II crystal structure. Finally, having outlined a general strategy for assigning backbone and side-chain resonances in a perdeuterated large protein, we propose a strategy whereby this information can be used to glean more detailed structural information from the partially or fully protonated protein equivalent.

Conformational preferences of a chimeric peptide HIV-1 immunogen from the C4-V3 domains of gp120 envelope protein of HIV-1 CAN0A based on solution NMR: comparison to a related immunogenic peptide from HIV-1 RF.

A critical problem to overcome on HIV vaccine design is the variability among HIV strains. One strategy to solve this problem is the construction of multicomponent immunogens reflective of common HIV motifs. Currently, it is not known if these motifs should be based primarily on amino acid sequence or higher-order structure of the viral proteins of a combination of the two. In this paper, we report NMR-derived solution conformations for a sympathetic peptide taken from the C4 and V3 domains of HIV-1 CAN0A gp120 envelope protein. This peptide, designated T1-SP10CAN0(A), is compared to a recently reported C4-V3 peptide. T1-SP10RF(A) from the HIV-1 RF strain [de Lorimier et al. (1994) Biochemistry 33, 2055-2062], in terms of conformational features and immune responses in mice [Haynes et al. (1995) AIDS Res. Hum. Retroviruses 11, 211-221]. The T1 segment of 16 amino acids from the gp120 C4 domain is identical in both peptides and exhibits nascent helical character. The SP10 region, taken from the gp120 V3 loop, differs from that of T1-SP10RF(A) in both sequence and conformations. A reverse turn is observed at the conserved GPGX sequence. The rest of the Sp10 domain is extended with the exception of the last three residues which show evidence for a helical arrangement. Modeling of the turn region of the T1-SP10CAN0(A) peptide shows exposure of a continuous apolar stretch of side chains similar to that reported in the crystal structure of a V3 peptide from HIV-1 MN complexed with a monoclonal antibody [Rini et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6325-6329]. this hydrophobic patch is interrupted by a charged Lys residue in the T1-SP10RF(A) peptide. This observation suggests that the HIV-1 CAN0A and HIV-1 RF C4-V3 peptides can induce widely different anti-HIV antibodies. consistent with immunogenic results.

Improved excitation pulse bandwidths using shaped pulses, with application to heteronuclear half filters in macromolecular NMR.

The advantageous use of sinc-shaped pulses in heteronuclear half filters is explored for studying biological macromolecules. The typical square, or hard, pulse used in half-filter pulse sequences for heteronuclear excitation results in suboptimal suppression of unwanted resonances due to incomplete inversion of spins. The novel use of short-duration shaped pulses applied at high power achieves more uniform excitation profiles over the extended frequency ranges often needed for heteronuclear filtering. This approach is used in the development of a double-tuned omega 1, omega 2-double-half-filtered, double-quantum-filtered COSY experiment. The efficiency of this experiment incorporating sinc pulses compares favorably with that obtained with square pulses in a mixture of 13C-labeled and unlabeled amino acids. Sinc-pulse-filtered spectra of the 24 kDa methionine repressor protein dimer MetJ, uniformly 13C-labeled expect at two unlabeled methionine residues, were also obtained to demonstrate the utility of this approach in biomacromolecular studies.

High-level 2H/13C/15N labeling of proteins for NMR studies.

The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with 2H, 13C and 15N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments, 2H has been incorporated into HCA II in order to decrease the rates of 13C and 1HN T2 relaxation. NMR quantities of protein with essentially complete aliphatic 2H incorporation have been obtained by growth of E. coli in defined media containing D2O, [1,2-13C2, 99%] sodium acetate, and [15N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for 13C and 15N backbone NMR assignment studies, since the 13C and 1HN T2 relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential 2H isotopic shift observed for partially deuterated CHnDm moieties.

Thermodynamic evaluation of binding interactions in the methionine repressor system of Escherichia coli using isothermal titration calorimetry.

The binding interactions of the methionine repressor protein, MetJ, from Escherichia coli with its cognate, metbox DNA sequence and corepressor S-adenosylmethionine were examined using calorimetric methods. A detailed thermodynamic characterization of this system which exhibits the recently reported (beta alpha alpha)2 binding motif provides values for delta G, delta H, and delta S for each step in the repressor binding cycle. These studies show that, in the presence of corepressor, MetJ binds to a single metbox operator site with delta G = -7.7 kcal.mol-1, whereas in the absence of corepressor, the free energy of interaction with a single site is -5.8 kcal.mol-1. Cooperative interactions between two repressor molecules bound to two adjacent sites contribute an additional free energy of -1.3 kcal.mol-1 to binding at the second site. Binding is enthalpically unfavorable in the absence of the corepressor with delta H = +2.6 kcal.mol-1 but becomes exothermic with delta H = -4.6 kcal.mol-1 when corepressor is present. The heat capacity for the system decreases significantly by delta Cp = -290 cal.mol-1.K-1 on a per site basis when the protein binds to DNA, and interactions between repressor molecules bound to adjacent sites contribute a delta Cp = -800 cal.mol-1.K-1, indicating that solvent exclusion plays a significant role in binding in this system. The corepressor binds to the unbound repressor protein with a free energy of delta G = -6.0 kcal.mol-1 and to the MetJ-operator complex with delta G = -6.95 kcal.mol-1. Repressor binding to random-sequence DNA was estimated to occur with a free energy of -5.7 kcal.mol-1 in the presence of corepressor. These data clearly indicate that MetJ repressor dimer binds specifically to the central region of its 8 bp cognate metbox operator but recognizes partial operator sequences as short as 6 bp. Cooperativity in binding of adjacent MetJ dimers to a double metbox sequence is demonstrated to be important in determining the energetics of the interaction. Finally, the corepressor S-adenosylmethionine enhances the affinity of MetJ for its recognition site DNA by a factor of 25 and contributes significantly to the net exothermicity of repressor binding.

NMR-derived solution conformations of a hybrid synthetic peptide containing multiple epitopes of envelope protein gp120 from the RF strain of human immunodeficiency virus.

Solution conformations of a 40-residue hybrid peptide containing T-helper epitopes and B-cell determinants from envelope glycoprotein gp120 of human immunodeficiency virus (HIV) have been investigated with NMR. Peptides of this general design are highly immunogenic and induce HIV-neutralizing antibodies and T-lymphocyte responses. The 16-residue N-terminal segment of the peptide contains a T-helper epitope, while the 24-residue C-terminal segment is derived from the V3 loop of HIV strain RF and contains epitopes that elicit neutralizing antibodies as well as T-cell responses. On the basis of 2D proton NMR spectra (COSY, TOCSY, and NOESY) of the peptide in aqueous solution, the resonances of nearly all hydrogens are assigned. The peptide is largely disordered, but specific medium-range NOEs demonstrate conformational preferences in certain regions. Part of the N-terminal segment exhibits nascent helical conformation, consistent with a finding that many T-cell antigens can be modeled as amphipathic helices. In the V3-derived segment of the peptide, one region shows evidence of a tight turn conformation, corresponding to a turn found previously in V3 peptides of HIV strains MN and IIIB. Other conformational features are also detected in the V3 region, such as a stretch of beta strand and a kink that may arise from side-chain interactions.

Proton NMR of Escherichia coli sulfite reductase: studies of the heme protein subunit with added ligands.

The heme protein subunit of sulfite reductase (SiR-HP; M(r) 64,000) from Escherichia coli as isolated contains the isobacteriochlorin siroheme exchange-coupled to a [4Fe-4S] cluster in the 2+ oxidation state. SiR-HP in the presence of a suitable electron donor can catalyze the six-electron reductions of sulfite to sulfide and nitrite to ammonia. Paramagnetic 1H NMR was used to study the low-spin complexes of SiR-HP formed by binding the exogenous inhibitor cyanide or the substrates sulfite and nitrite. As a model, the cyanide complex of purified siroheme was also prepared. The NMR spectrum of isolated ferric low-spin siroheme-CN is consistent with spin density being transferred into the a2u molecular orbital, an interaction which is symmetry-forbidden in porphyrins. The pattern of proton NMR shifts observed for isolated ferric low-spin siroheme-CN is very similar to those obtained for the protein-cyanide complex. NMR spectra of the cyanide complex of SiR-HP were obtained in all three accessible redox states. The pattern of hyperfine shifts observed for the one-electron and two-electron reduced cyanide complexes is typical of those seen for [4Fe-4S] clusters in the 2+ and 1+ oxidation states, respectively. Resonances arising from the beta-CH2 protons of cluster cysteines have been assigned for all complexes studied utilizing deuterium substitution. The cyanide-, sulfite-, and nitrite-ligated states possessed an almost identically shifted upfield cluster cysteine resonance whose presence indicates that covalent coupling exists between siroheme and cluster in solution. Data are also presented for the existence of a secondary anion binding site, the occupancy of which perturbs the oxidized SiR-HP NMR spectrum, where binding occurs at a rate much faster than that of ligand binding to heme.

Proton NMR of Escherichia coli sulfite reductase: the unligated hemeprotein subunit.

The isolated hemeprotein subunit of sulfite reductase (SiR-HP) from Escherichia coli consists of a high spin ferric isobacteriochlorin (siroheme) coupled to a diamagnetic [4Fe-4S]2+ cluster. When supplied with an artificial electron donor, such as methyl viologen cation radical, SiR-HP can catalyze the six electron reductions of sulfite to sulfide and nitrite to ammonia. Thus, the hemeprotein subunit appears to represent the minimal protein structure required for multielectron reductase activity. Proton magnetic resonance spectra are reported for the first time on unligated SiR-HP at 300 MHz in all three redox states. The NMR spectrum of high spin ferric siroheme at pH 6.0 was obtained for the purpose of comparing its spectrum with that of oxidized SiR-HP. On the basis of line widths, T1 measurements, and 1D NOE experiments, preliminary assignments have been made for the oxidized enzyme in solution. The pH profile of oxidized SiR-HP is unusual in that a single resonance shows a 9 ppm shift over a range of only 3 pH units with an apparent pK = 6.7 +/- 0.2. Resonances arising from the beta-CH2 protons of cluster cysteines have been assigned using deuterium substitution for all redox states. One beta-CH2 resonance has been tentatively assigned to the bridging cysteine on the basis of chemical shift, T1, line width, and the presence of NOEs to protons from the siroheme ring. The observed pattern of hyperfine shifts can be used as a probe to measure the degree of coupling between siroheme and cluster in solution. The cluster iron sites of the resting (oxidized) enzyme are found to possess both positive and negative spin density which is in good agreement with Mossbauer results on frozen enzyme. The NMR spectrum of the 1-electron reduced form of SiR-HP is consistent with an intermediate spin (S = 1) siroheme. Intermediate spin Fe(II) hemes have only been previously observed in 4-coordinate model compounds. However, the amount of electron density transferred to the cluster, as measured by the isotropic shift of beta-CH2 resonances, is comparable to that present in the fully oxidized enzyme despite diminution of the total amount of unpaired spin density available. Addition of a second electron to SiR-HP, besides generating a reduced S = 1/2 cluster with both upfield and downfield shifted cysteine resonances, converts siroheme to the high spin (S = 2) ferrous state.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphorus 31 magnetic resonance spectroscopy of perifused human placental villi under varying oxygen concentrations.

OBJECTIVE: Initial phosphorus magnetic resonance spectroscopy observations on the oxygen metabolism of placental villi from normal term pregnancies are described. STUDY DESIGN: Villi were suspended in medium and perifused within a custom-designed 30 mm nuclear magnetic resonance probe in a superconducting vertical nuclear magnetic resonance magnet where pH, temperature, and oxygenation were monitored. RESULTS: Phosphorus resonances were observed from adenosine triphosphate, phosphomonoesters. inorganic phosphate, and phosphodiesters. No phosphocreatine signal was observed. The placental villus tissue responded to an increase in oxygen concentration of the perifusate with a rise in the adenosine triphosphate level and a concomitant decline in the inorganic phosphate and the phosphomonoester signals. CONCLUSION: The changes observed reflect continuing dynamic glycolysis and oxidative phosphorylation. The absence of a phosphocreatine peak suggests that aerobic pathways not driven by creatine kinase are important for placental metabolism. Our system demonstrates dynamic oxygen metabolism in perifused viable placental villus tissue by means of magnetic resonance spectroscopy.

Comparative analysis of normal and growth-retarded placentas with phosphorus nuclear magnetic resonance spectroscopy.

OBJECTIVE: Phosphorus 31 magnetic resonance spectroscopy studies were carried out on placentas from normal vaginal and elective cesarean deliveries without antenatal complications and from pregnancies complicated by intrauterine growth retardation of unknown cause to determine differences. STUDY DESIGN: Perchloric acid extraction was performed on frozen tissue, and quantitative analysis was carried out for well-resolved resonances representing adenosine triphosphate, sugar phosphate, inorganic phosphorus, diphosphoglycerate, glycerophosphorylethanolamine, and glycerophosphorylcholine. RESULTS: Adenosine triphosphate levels were highest in the growth-retarded group. There were significantly higher levels of sugar phosphate, diphosphoglycerate, and glycerophosphorylcholine in the placentas of the growth-retarded pregnancies compared with those from normal placentas. CONCLUSION: These differences may represent a response to hypoxia and an increase in the amount of blood in the placenta. The results demonstrate the utility of nuclear magnetic resonance spectroscopy for studying the pathology of abnormal placentas to gain a better understanding of the pathology and represent early steps toward in vivo spectroscopic studies of the placenta.

A refocused and optimized HNCA: increased sensitivity and resolution in large macromolecules.

A 3D optimized, refocused HNCA experiment is described. It is demonstrated to yield a dramatic increase in sensitivity when applied to [13C, 15N]-labeled human carbonic anhydrase II, a 29-kDa protein. The reasons for the gain in sensitivity are discussed, and 3 distinct areas for further development are indicated.

Characterization of a microcarrier cell culture system for 23Na MR spectroscopy studies.

A MR spectroscopy method is described for the simultaneous discrimination and observation of sodium from the three compartments created by an intact cell monolayer. Results are reported for Madin Darby Canine Kidney (MDCK) cells, an epithelial-like continuous cell line, cultured on Cytodex 1 microcarrier beads and perfused with medium containing 6 mM dysprosium (III) tripolyphosphate [Dy(TPP)2(7-)] as shift reagent. The sodium spectrum shows three resonances which are assigned to the shifted intrabead (basolateral) and extrabead (apical) pools and the unshifted intracellular pool. Ouabain inhibition of the Na(+)-K(+)-ATPase cellular pump mechanism was used to demonstrate the sensitivity of the method for monitoring intracellular sodium. The supported MDCK cells in this system remained viable after exposure for 5 h to medium containing Dy(TPP)2(7-) at a concentration of 6 mM, as determined by trypan blue dye exclusion and by comparison of the log growth rate and ability to form domes in subsequent generations of exposed cells vs unexposed controls.

Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II.

Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of [13C6, 99%]glucose. We demonstrate here that uniformly (greater than 95%) 13C and 15N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium [1,2-13C2, 99%]acetate as the sole carbon source and [15N, 99%]ammonium chloride as the sole nitrogen source. In addition, we demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium [1-13C, 99%]acetate and sodium [2-13C, 99%]acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the 1H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium [1,2-13C2, 99%]acetate growth media is considerably less than the cost of the [13C6, 99%]glucose growth media. We report here the first published 13C and 15N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.

Phosphorus 31 magnetic resonance spectroscopy of human placenta and quantitation with perchloric acid extracts.

Phosphorus 31 magnetic resonance spectroscopic studies of fresh placental tissue are reported that indicate resonances for adenosine triphosphate, inorganic phosphate, sugar phosphates-phosphomonoesters, and phosphodiesters. Perchloric acid extract methods were used to further characterize and quantitate phosphorous metabolites in term human placentas by phosphorus 31 magnetic resonance spectroscopy. The perchloric acid extracts give enhanced resolution of phosphorus signals and allow identification of other phosphorus metabolites including small amounts of phosphocreatine. Emphasis was placed on quantitating adenosine triphosphate levels in the acid extracts with the use of the external reference standard hexachlorocyclotriphosphazene in a coaxial capillary system. Adenosine triphosphate levels measured in this way ranged from 0.404 to 0.709 mumol per gram wet weight. Comparison with an internal standard method with phosphocreatine is also reported. Contribution to the measured high-energy phosphate pool from blood in the highly vascularized tissue was found to be relatively large and could range from 30% to 50% of the total adenosine triphosphate measured.

Use of gel filtration in the preparation of biological fluids for magnetic resonance spectroscopy.

Analysis of biological fluids by proton magnetic resonance spectroscopy is often complicated by dynamic range problems created from the large water resonance. Gel filtration chromatography is found to be a simple and nondestructive method for exchanging D2O for H2O and for removing low molecular weight molecules from both plasma and urine, significantly improving subsequent one- and two-dimensional MRS spectra.

Synthesis of 11C-labeled chlorpromazine directly from [11C]carbon dioxide.

Radiolabeled chlorpromazine was prepared by carboxylation of the N-trimethylsilyl derivative of norchlorpromazine with [11C]carbon dioxide, followed by in situ lithium aluminum hydride reduction. Radiochemical yields of 22-24% and radiochemical purities in the range of 93-98% were achieved.