Disaccharide analysis of heparin and heparan sulfate using deaminative cleavage with nitrous acid and subsequent labeling with paranitrophenyl hydrazine.

Compositional analyses of heparin (Hep) and heparan sulfate (HS) have been undertaken with disaccharide units obtained by either enzymatic digestion with heparitinases or hydrazinolysis/deamination reaction of polysaccharides. Unsaturated disaccharide units generated by the enzymatic method are detectable on HPLC with a uv detector recording absorbance at 230 nm. On the other hand, disaccharide units generated by the chemical method possess a component of 2,5-anhydromannose (AnMan) bearing aldehyde groups in addition to intact iduronic acid (IdoA) or glucuronic acid (GlcA). The aldehyde groups of the disaccharide units are usually reduced with sodium borotritide, and detected by radiochromatography. Both of them, however, involve inevitable experimental problems, such as the use of costly enzymes and radioisotopes. In the present study, we have established a novel composition analysis system for Hep and HS essentially based on the chemical method. After hydrazinolysis/deamination treatment of Hep and HS, the aldehyde groups of AnMan in the disaccharide units generated were coupled with paranitrophenyl (PNP-) hydrazine instead of reduction with sodium borotritide, AnMan-CH=N-NH-PNP (AnMan-PNP) being formed. Then, the PNP-labeled disaccharides were pre-treated on a SepPak C-18 cartridge column, and subsequently separated and detected on ion-pairing reversed-phase HPLC with a detector recording absorbance at 390 nm. With the present system, the order of elution was GlcA-AnMan-PNP (GM), IdoA-AnMan-PNP (IM), IdoA(2S)-AnMan-PNP (ISM), IdoA-AnMan(6S)-PNP (IMS), and IdoA(2S)-AnMan(6S)-PNP (ISMS). As an application, the disaccharide compositions of heparin from bovine intestine and heparan sulfate from bovine kidney were analyzed by the present method, and the results were comparable to those obtained by a well-established enzymatic method. The present compositional analysis was demonstrated to be reliable and economical.