N.m.r. studies of metabolism in perfused organs

Several metabolites and intracellular pH in intact organs can be studied in a non-destructive manner by phorphorus nuclear magnetic resonance (31P n.m.r.). This possibility was demonstrated by us nearly five years ago. Since then we have developed the appropriate physiological techniques and improved the n.m.r. method for the study of animal hearts and kidneys. Here we described measurements aimed at clarifying three problesm. (1) Having measured the enzyme-catalysed fluxes between phosphocreatine and ATP by the method of saturation transfer n.m.r., we examine the relations between energy supply and heart rate in the isolated perfused rat heart. (2) We describe experiments to establish the validity of the perfusion model. For the first time, we report 31P n.m.r. measurements of an in vivo rat heart and compare the results with those obtained for the perfused rat heart. (3) Ischaemia and metabolism in rabbit kidneys is investigated to establish the relation between functional and metabolic recovery after a renal transplant operation.     

N.m.r studies of internal mobility in a cyclic tetrapeptide

The conformation of cyclo(D-Phe-D-Pro-Ala-Pro) is reported. Measurements of spin-lattice relaxation in the rotating frame indicate that this peptide is conformationally less mobile on the microsecond time scale than larger cyclic peptides previously studied. Libration of the Pro-Ala and Pro-Phe peptide bond planes is suggested as the source of the small exchange contributions to 1/T1p.     

N.m.r. and biochemistry

The role and historical progress of n.m.r. applications in biochemistry are briefly outlined. Technical advances over the years have made n.m.r., at last, a technique which can give valuable information about a wide range of biochemical topics, from enzyme kinetics in vivo to the structure of protein-DNA complexes. Emphasis here is placed on studies of proteins, especially those made up from mosaics of modules. It is shown that n.m.r. can readily give detailed structural information about individual protein modules and that valuable information about the structure and function of the intact mosaic protein can be inferred.     

N.m.r. studies of cycloamylose inclusion-complexes with p-substituted phenols

Aqueous solutions of inclusion complexes of host cyclomaltohexa- and cyclomaltohepta-ose (cyclohexa- and cyclohepta-amylose) with guest phenols p-substituted with Br, Cl, CN, NO₂, CH₃, CHO, and COOH have been studied by ¹H- and ¹³C-n.m.r. spectroscopy. The stability of the complexes depends on the guests, the cycloamyloses, and the ionisation states of the host hydroxyl groups. The ¹H data for the host protons in the complexes show that the guests are more deeply inserted into the cavity of cyclomaltoheptaose than into that of cyclomaltohexaose.     

N.m.r. studies of synergistic kappa carrageenan—carob galactomannan gels

¹³C-n.m.r. spectroscopy has been used to investigate the carob galactomannan—kappa carrageenan binary gels. Starting from partially depolymerized carob samples, evidence for interaction and intermolecular binding was found by analysis of the spectra obtained in quantitative conditions and in the absolute intensity mode. From these, a reconstitution of the signals corresponding to C-1, C-4, C-5 and C-6 showed the existence of three kinds of galactomannan chains: the first ones with a low galactose content were strongly connected and as much had completely lost their mobility; but the chains with a high galactose content were still detected and could be divided in two groups according to their mobility.     

Glucosamine oligomers: 2. N.m.r. studies on a DP3.

This paper concerns the characterization of the chemical structure of a DP3 glucosamine oligomer. The assignments of nearly all protons are reported. Variations of 1H chemical shifts with pD and temperature are correlated to the pKa of amino groups, and not with substantial conformational changes.     

N.m.r. imaging of intact biological systems

Internal images of structured objects may be obtained with n.m.r. by labelling component parts with different magnetic field strengths and therefore recognizably different n.m.r. frequencies. A linear field gradient generates a one-dimensional projection of nuclear density and a variety of techniques are employed to manipulate this one-dimensional probe to yield internal images in two and three dimensions. In the past few years, n.m.r. imaging, sometimes also called zeugmatography or spin mapping, has been applied progressively to provide proton images of small phantoms, fruit, vegetables and small animals, and finally to in vivo imaging of the human body; it promises to provide a valuable means of interior investigation of intact biological systems generally. For medical imaging the method is non-invasive, does not use ionizing radiations, appears to be without hazard and penetrates bony cavities without attenuation. Furthermore, other n.m.r. parameters, for example, relaxation times and fluid flow, may also be mapped; there is evidence that the relaxation times from tumours are significantly longer than those from corresponding normal tissue. Effort to date has mostly been concentrated on proton n.m.r., but some work has been done with other nuclei. Three examples are shown of n.m.r. images of intact biological systems: a fruit, an animal and a human system. The discussion includes the quantitative nature of the images, tissue discrimination, the relation between the resolution in the image and image acquisition time, attenuation and phase shift of the r.f. field in the biological tissue, and magnets suitable for n.m.r. imaging. In principle, all conventional n.m.r. techniques can be combined with n.m.r. methods in order to investigate heterogeneous systems. Overhauser imaging is briefly discussed.     

N.m.r. studies of the disulphated disaccharide obtained by degradation of bovine lung heparin with nitrous acid

The disulphated disaccharide IdoA(2SO3)-anManOH(6SO3) was prepared from bovine lung heparin by treatment with nitrous acid followed by borohydride reduction. The 1H- (400 MHz) and 13C-n.m.r. (100 MHz) spectra of this disaccharide derivative have been assigned completely using homonuclear spin-decoupling experiments, 13C-1H correlations, and a COSY-45 two-dimensional homonuclear correlation experiment. The 3JH,H values show that the IdoA(2SO3) residue exists in a single conformation throughout the temperature range 20-90 degrees.     

1H-n.m.r. studies of squash seed trypsin inhibitor

1H-n.m.r. studies at 500 MHz have been performed on a trypsin inhibitor (CMTI-III) found in squash seed (Cucurbita maxima). The sequential resonance assignments have been made using two-dimensional techniques. The chemical shifts for the assigned protons are reported at 30 degrees, pH 2.8 and form a basis for the determination of the solution structure of CMTI-III. Analysis of the NOE data, NH-alpha CH vicinal coupling constants and pattern of slowly exchanging amide protons indicates that the predominant feature of the solution conformation is a triple stranded beta sheet consisting of residues 8-10, 21-23, and 26-29. Residues 12-15 appear to form a beta turn.     

N.m.r. relaxation and images of human breast tumours in vitro

It is found that fat and non-fatty tissue in dissected samples of the mamma differ in their T1/T2 ratios. This opens the possibility of locating tumours by n.m.r. imaging, because they have a lower fat content than their surroundings. By means of a sensitive point method, samples were scanned with a resolution of about 0.4 mm X 0.4 mm. The similarity between the shape of a tumour in an n.m.r. and in an X-ray image of a thin section of mamma tissue is quite convincing.     

N.m.r. studies of d-ribosylamines in solution: Derivatives of hydroxylamine,hydrazine, thiosemicarbazide,and secondary amines

N.m.r. spectroscopic studies (¹H, ¹³C) have shown that hydroxylamine and hydrazine react with 2,3-O-isopropylidene-d-ribofuranose (1) and d-ribose (2) to give primarily the acyclic oxime and hydrazone, respectively, whereas thiosemicarbazide affords mainly the cyclic pyranosyl and furanosyl derivatives. Acyclic or cyclic secondary amines, when condensed with either 1 or 2, furnished only mixtures of the β-pyranosyl and β-furansyl forms, and, in some reactions, Amadori rearrangement products (2–30%).     

N.m.r. studies of myelin basic protein. Conformation of a peptide that is an antigenic determinant for B-cell reactivity.

The peptide Gly-Arg-Ala-Ser-Asp-Tyr-Lys-Ser, derived from myelin basic protein (MBP), is part of an epitope to monoclonal antibodies to human MBP. Its conformation has been studied in aqueous solution by high-resolution one- and two-dimensional 1H and 13C n.m.r. Two-dimensional correlated spectroscopy, pH titrations and one-dimensional spin-decoupling techniques were employed to assign the spectra observed from both nuclei. Amide proton temperature coefficients, coupling constants, 13C spin-lattice relaxation times and nuclear-Overhauser-effect data provide evidence that the solution conformations of the octapeptide include a type-II beta-turn with a hydrogen bond between the CO group of Arg2 and the NH group of Asp5. The results are discussed in view of a possible conformation of the antibody receptor site.