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N-Glycosidically bound sialooligosaccharides in a model glycoprotein of serum type (porcine thyroglobulin) were released by pronase digestion, followed by hydrazinolysis. The resulting oligosaccharides were re-N-acetylated and reduced with sodium borohydride. On the other hand, O-glycosidically bound sialooligosaccharides were released from a mucin-type glycoprotein (bovine submaxillary mucin) with alkali in the presence of sodium borohydride. The reduced oligosaccharides thus obtained from both types of glycoproteins were analysed by high-performance liquid chromatography on a column of a latex-type pellicular anion-exchange resin with strong alkali as eluent. These sequential procedures were useful for mapping of oligosaccharides in glycoproteins.

A post-column derivatization method for the high-performance liquid chromatography of peptides and proteins giving a fluorescence intensity proportional to the number of peptide bonds is described. Peptide bonds were chlorinated with hypochlorite and the N-chlorite formed was allowed to react with thiamine to give fluorescent thiochrome. This method was applied the determination of membrane-forming proteins of microorganisms.

The effects of storage of size-exclusion column packing materials in methanolic or azide-water solutions on subsequent separations were tested. Three commercially available columns were used in these studies; the Toyo-Soda Bio-Sil TSK 125, Bio-Sil TSK 250 and the DuPont Bio-Series GF-250. Upon initial chromatography, all three columns bound up to 760 micrograms of cytochrome c tryptic peptides. Sample binding to packing material is probably a function of the positively charged basic groups on peptides or proteins interacting with silanol groups. The larger the peptide, the less the opportunity for silanol-charged group interaction, hence, less binding. Initial samples introduced to a new column occupy the binding sites. Equilibration with neat methanol removes the bound protein revealing sites which bind sample. After absorption of peptides to binding sites on the packing material, storage in neat methanol regenerates the binding sites. Storage in 10% methanol diminished the binding phenomenon, but storage in azide-water reduced binding to a range below detection at the microgram level. Our recommendation to users of size-exclusion chromatographic columns is that one satisfy the absorption capacity of a new column by injecting a sufficient quantity of a basic peptide standard or other convenient sample to reduce available binding sites before using the column for important separations. Store columns in azide-water or 10% methanol to prevent the regeneration of exposed silanol groups.

Thyroid peroxidase and lactoperoxidase are capable of producing oxidized bromine species. Thus investigations into bromination reactions with tyrosine and thyroglobulin were undertaken in order to gain insight into possible formation of brominated thyroid hormone analogues. A reversed-phase high-performance liquid chromatographic method was developed for the separation of bromine/iodine-substituted tyrosines and used as a basis for these investigations combined with ultraviolet absorption and electrochemical detection. The results indicate that in vivo bromination of tyrosyl residues in thyroglobulin might be of some importance in cases of either iodine deficiency or excessive bromide intake.

When peptides containing thyroid hormones are first solubilized in 6 M guanidine hydrochloride they can be perfectly separated by reversed-phase high-performance liquid chromatography on an octadecylsilica column using conventional elution conditions (trifluoroacetic acid-acetonitrile).