Proximate analysis is often used in decomposition studies to characterize the organic components of foliar litter. The percent weight residue remaining after extraction by nonpolar and polar solvents and H3SO4 hydrolysis (Klason lignin, KL) is commonly used as a measure of litter quality and a modelling parameter. While KL is associated with resistance to decay, its nature is not well understood and it has long been suspected that it incorporates nonlignin components. We used solid-state 13C nuclear magnetic resonance (NMR) spectroscopy to characterize litter, extracted residue, and the KL fraction of five species. NMR shows that cutin and condensed tannin are both significant components of litter and its extraction residues, in addition to lignin and carbohydrate. Hydrolysis with H2SO4 removes carbohydrates and amino acids, leaving the KL fraction derived from cutin, tannin, and lignin. Tannin retention in KL was also demonstrated by a hydrolysis study of purified tannins and a brown-rot lignin, using both NMR and the proanthocyanidin assay for condensed tannins. Although the NMR results are qualitative at this stage, it is clear that KL has limited use as a parameter controlling litter decomposition, and that other biopolymers should not be ignored in conceptual models, chemical analysis, and experimental design. Key words: Klason lignin, 13C CPMAS NMR, proximate analysis, litter quality, decomposition, tannin.

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A sample preparation method using spherical glass ampoules has been used to achieve 1.5-Hz resolution in 1H magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of aqueous multilamellar dispersions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), serving to differentiate between slowly exchanging interlamellar and bulk water and to reveal new molecular-level information about hydration phenomena in these model biological membranes. The average numbers of interlamellar water molecules in multilamellar vesicles (MLVs) of DOPC and POPC were found to be 37.5 +/- 1 and 37.2 +/- 1, respectively, at a spinning speed of 3 kHz. Even at speeds as high as 9 kHz, the number of interlamellar waters remained as high as 31, arguing against dehydration effects for DOPC and POPC. Both homonuclear and heteronuclear nuclear Overhauser enhancement spectroscopy (NOESY and HOESY) were used to establish the location of water near the headgroup of a PC bilayer. 1H NMR comparisons of DOPC with a lipid that can hydrogen bond (monomethyldioleoylphosphatidylethanolamine, MeDOPE) showed the following trends: 1) the interlamellar water resonance was shifted to lower frequency for DOPC but to higher frequency for MeDOPE, 2) the chemical shift variation with temperature for interlamellar water was less than that of bulk water for MeDOPE MLVs, 3) water exchange between the two lipids was rapid on the NMR time scale if they were mixed in the same bilayer, 4) water exchange was slow if they were present in separate MLVs, and 5) exchange between bulk and interlamellar water was found by two-dimensional exchange experiments to be slow, and the exchange rate should be less than 157 Hz. These results illustrate the utility of ultra-high-resolution 1H MAS NMR for determining the nature and extent of lipid hydration as well as the arrangement of nuclei at the membrane/water interface.

Influenza virus hemagglutinin (HA)-mediated membrane fusion involves insertion into target membranes of a stretch of amino acids located at the N-terminus of the HA(2) subunit of HA at low pH. The pK(a) of the alpha-amino group of (1)Gly of the fusion peptide was measured using (15)N NMR. The pK(a) of this group was found to be 8.69 in the presence of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The high value of this pK(a) is indicative of stabilization of the protonated form of the amine group through noncovalent interactions. The shift reagent Pr(3+) had large effects on the (15)N resonance from the alpha-amino group of Gly(1) of the fusion peptide in DOPC vesicles, indicating that the terminal amino group was exposed to the bulk solvent, even at low pH. Furthermore, electron paramagnetic resonance studies on the fusion peptide region of spin-labeled derivatives of a larger HA construct are consistent with the N-terminus of this peptide being at the depth of the phosphate headgroups. We conclude that at both neutral and acidic pH, the N-terminal of the fusion peptide is close to the aqueous phase and is protonated. Thus neither a change in the state of ionization nor a significant increase in membrane insertion of this group is associated with increased fusogenicity at low pH.

13C cross-polarization magic-angle spinning nuclear magnetic resonance (CPMAS NMR) spectroscopy was used to characterize heartwood from decaying fallen boles of Douglas-fir (Pseudotsugamenziesii (Mirb.) Franco), western hemlock (Tsugaheterophylla (Raf.) Sarg.), and western red cedar (Thujaplicata Donn). The sample decay classes I to V had been previously assigned based on field observations. Solid-state 13C CPMAS NMR spectra were analyzed to determine the proportion of C of the following chemical types: carbohydrate, lignin, aliphatic, and the sum of carboxyl plus carbonyl. For both Douglas-fir and western hemlock, the proportion of carbohydrate C increased slightly in the early stages of decay. This was followed by a substantial increase in lignin C, while carbohydrate C declined to about 10% of total C. By contrast, the spectra for western red cedar generally showed little change with increasing decay class. One exceptional sample of western red cedar class IV was highly decomposed, indicating complete loss of carbohydrate C, and some loss of lignin side-chain C. For all three species, signals from alkyl and carbonyl C were weak, but tended to increase slightly with decomposition, most likely because of the selective preservation of waxes and resins (alkyl C), and oxidation. Accumulation of chitin was not observed, and there was little evidence for lignin decomposition or for formation of humic polymers. 13C CPMAS NMR offers a simple and information-rich alternative to wet chemical analyses to monitor changes in organic components during decomposition of woody litter.