The extracellular hemoglobin of Ascaris has an extremely high oxygen affinity (P5. = 0.004 mmflg). All retain the heme-linked proximal histidine (F8) and the phenylalanine (CD1) that wedges the heme into its pocket. Most of them also show the distal histidine (E7) and valine or isoleucine (Ell), but more important, all retain a pattern ofabout 33 internal sites from which all polar residues except serine and threonine residues are excluded (6, 7). Ascaris is a parasitic nematode that lives in the intestines of animals where oxygen is scarce. It contains a hemoglobin with an oxygen affinity two orders of magnitude higher than that of any vertebrate myoglobin or hemoglobin or any synthetic iron porphyrin made so far (P50 = 0.004 mmHg). We know of no way by which the oxygen affinity offerrous heme could be raised to that level. As a first step toward the solution of this chemical mystery, we have determined the amino acid sequence of Ascaris hemoglobin.tt MATERIALS AND METHODSThe perienteric fluid hemoglobin of Ascaris suum, obtained in a local slaughterhouse, was prepared essentially according to ref. 8. The final preparation, which was passed over a Superose 6 column in 50 mM Tris HC1, pH 7.5/100 mM NaCl, had an A412/A28w = 1.3 and showed a single but broad spot on two-dimensional SDS/PAGE. The protein was split chemically or enzymatically. Chemical cleavage was done by CNBr, 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine, or partial acid hydrolysis (9). Enzymatic cleavage was done on the denatured, unmodified maleylated protein, by either trypsin, chymotrypsin, or Staphylococcus V8 protease. Conformation-dependent cleavage was done with subtilisin (9). Specific cleavage after lysine was done with endoproteinase Lys-C (10). Analysis with carboxypeptidase P was done at pH 5.6 and 250C with an enzyme-to-substrate ratio of 1:200. Peptide mixtures from chemical or enzymatic cleavage were separated by reversed-phase HPLC on C4 (Vydac) or C18 (uBondapak; Waters) columns, and the peptides were eluted with acetonitrile in 0.1% trifluoroacetic acid at or near neutral conditions (50 mM triethylamine/acetic acid, pH 6.5). Several CNBr peptides were purified by preparative SDS/PAGE and electroelution according to refs. 11 and 12. Coomassie stain was displaced from the peptide with 1% SDS and extracted with four volumes of acetone.

The primary structures of 21 novel monoantennary and diantennary N-glycans of the glycoprotein a,-hemocyanin (a,-Hc) of Helix pnmutia have been determined. Outer oligosaccharide fragments (antennae) were released from the glycoprotein by Smith degradation of an a,-Hc pronase digest. The major antenna, obtained following HPLC fractionation on Lichrosorb-NH,, was characterized using 'H-NMR spectroscopy, fast-atom-bombardment mass spectrometry, and linkage analysis, and corresponds to a pentasaccharide fragment. The intact carbohydrate chains of a,,-Hc were released with peptide-N4-(Nacetyl-P-glucosaminy1)asparagine amidase-F digestion, separated from the protein on Bio-Gel P-100, and subfractionated on Bio-Gel P-4. A portion of subfractions was reduced with sodium borodeuteride, and the non-reduced and reduced samples were further fractionated on CarboPac PA-1, Lichrosorb-NH,/ Lichrosphere-NH2, and/or Lichrosphere-C,,. Purified oligosaccharides and oligosaccharide-alditols were analyzed using 500/600-MHz 'H-NMR spectroscopy. In total, four novel types of antenna were identified, namely, 3MeGalwl-6)\ \ \ 3MeGal(~1-3)GalNAc(~1-4)GlcNAc(~1-4MeGal(~I-3)GalNAc(/j'1-4)GlcNAc(~l-which are all attached to 0 -2 of aMan residues of the trimannosyl-N,N'-diacetylchitobiose core element, which is generally P-1,2-xylosylated and a-I ,6-fucosylated, ][? Xyl(p1-2)]Man(jl1-4)GlcNAc(~1-4) [ 2 Fuc( a 1 -6)] GlcNAc.

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