The N-acetyl-beta-D-hexosaminidase B of germinating Lupinus luteus L. seeds (McFarlane et al. (1984) Phytochemistry 23, 2431-2433) was partially purified with a six-step purification procedure following extraction. This enzyme consists of one protein chain (Mr 69,000, as determined by SDS-PAGE and 62,500, as obtained by gel filtration on Bio-Gel P-60 Gel) and has a neutral isoelectric point (pI = 7.05, as determined by chromatofocusing). Moreover, it was found to be very sensitive to low ionic strength, especially in the presence of different gels based on Sephadex. Considering the substrate specificity, the enzyme splits both p-nitrophenyl-2-acetamido-2-deoxy-beta-D-glucosaminide and -galactosaminide substrates, but lacks N,N'-diacetylchitobiase activity. A new mixed-substrate procedure was developed and is presented here to demonstrate that a common active site is responsible for the splitting of both synthetic substrates.
