The quantitation of glucosamine and galactosamine in biological samples can be done simply and accurately on an amino acid analyzer (l-3). However, a problem often arises from the presence of tryptophan or its degradation products or from peptides produced during the mild acid hydrolysis required for amino sugar liberation.
Fanger and Smyth (4) reported the use of a column of Dowex 2-X4 to remove neutral and acidic amino acids and peptides. Amino sugars were eluted with a volatile buffer and were subsequently separated on a 20-cm column on the amino acid analyzer.
We have developed a more simple, alternate procedure to analyze amino sugars in the presence of peptides and tryptophan using a short column of the amino acid analyzer equilibrated in a low-molarity buffer. This system also separates glucosamine from glucosaminitol.
Samples were prepared by hydrolysis of 0.25 to 0.5 mg of protein with 1 ml of 2 N HCl in heavy-walled ignition tubes. The tubes were alternately evacuated and flushed with prepurified nitrogen four times before the final evacuation and hydrolysis at 110Β°C for 12 hr. The tubes were opened and rinsed with 8 ml of deionized, distilled water into lOO-ml round-bottom flasks, and the samples were concentrated to dryness by rotary evaporation at 40Β°C in VCICUO. The samples were rinsed twice with water (5.0 ml) and dried and subsequently taken up in 1.0 ml of 0.2 M sodium citrate, pH 2.2 (5). The amino acid calibration mixture was purchased from Beckman Instruments, Inc. Other amino sugar and tryptophan standards were prepared as 2.5 ~mollml in 0.01 N HCl. Glucosaminitol was a gift of Dr. F. Maley and rabbit IgG and Fab were gifts of Dr. K. Amiraian, of this division. Suitable dilutions in 0.2 M sodium citrate, pH 2.2, were used.
Analyses were performed on an amino acid analyzer (3) modified for accelerated chromatography (6) and for the use of the dimethyl sulfoxide reagent (7). Samples were applied to a column of PA-35 resin (Beckman Instruments, Inc.), 0.9 x 10.5 cm, through a manual sample injector valve containing a 0.80-ml sample loop (Beckman Instruments, Inc.). The column was maintained at 52Β°C. Flow rates of 60 and 35 ml/hr for buffer and ninhydrin solutions, respectively, were used.
The pH 5.28 citrate buffer, 0.35~ in sodium, was prepared according 532 Copyright