Human xylosyltransferases I and II (XyIT-I, XyIT-II) are key enzymes in glycosaminoglycan biosynthesis. Knowledge about the in vivo molecular weight, oligomeric state or turnover number are essential characteristics which have been addressed in this study. XyIT-II was purified from Pichia pastoris by fractionated ammonium sulfate precipitation, heparin affinity and ion exchange chromatography. XyIT-II was purified over 7000-fold with a final yield of 2.6%. By utilizing mass spectra analysis we can prove its first in-gel detection showing a migration pattern behavior that confirms its in silico, molecular weight of 95.8 kDa. We could determine a turnover number of 2.18 min (1) or one transferred xylose molecule per one XyIT-II Molecule each 27.5 s. The k(cat)/K-M ratio was 0.357 min (1) mu M (1) for XyIT-I using the bikunin-homologous acceptor Bio-QEEEGSGGGQKK-F. The comparison to XyIT-I derived from the same organism revealed a 2.4-fold higher catalytic efficiency (0.870 min (1) mu M (1)) for XyIT-I. (C) 2008 Elsevier Inc. All rights reserved.