Biochemical and Immunochemical Characterization of Hexosaminidase Pt

Hexosaminidase P, the main isozyme of hexosaminidase in pregnancy serum, was isolated and purified 600-700-fold by a two-step purification procedure-affinity chromatography on Sepharose-bound eaminocaproyl-Nacetylglucosylamine, followed by ion-exchange chromatography on DEAE-cellulose. The purified enzyme was subjected to biochemical and immunochemical analysis. Its catalytic property, namely, kinetic behavior, is similar to that of the major isozymes of hexosaminidase, A and B. However, it differs from these isozymes in its electrophoretic mobility and in T h e enzyme N-acetyl-P-D-hexosaminidase (hexosaminidase, EC 3.2.1.30) is present in human tissues as two main isoenzymes designated hexosaminidase A and B (Robinson & Stirling, 1968) which are localized in the lysosomal fraction of the cells. However, analysis of the enzyme obtained from different tissues, cells, or body fluids indicated the existence of some minor hexosaminidase isozymic forms such as hexosaminidase C (Poenaru et al., 1973; Braidman et al., 1974a,b; Penton et al., 1975; Reuser & Goljaard, 1976), 11 and 12 (Price & Dance, 1972), etc. Another isoenzyme was detected in sera of pregnant women (Stirling, 1971, 1972) and was denoted hexosaminidase P. The increase in levels of hexosaminidase activity in serum during pregnancy was first observed by Walker et al. (1960). In later reports O'Brien et al. (1 970) and Huddlestone et al. (1971) suggested that this elevation in activity is due to an increased hexosaminidase B level. However, later studies have demonstrated that the increase in enzymatic activity is due to the appearance of a novel enzyme in the serum, namely, hexosaminidase P. During the last few years remarkable progress has been made in the elucidation of the molecular structure of the two major human isozymes, hexosaminidases A and B. Based on experimental evidence concerning various aspects of the enzyme as derived from biochemical studies, somatic cell hybrids (Lalley et al., 1974; Gilbert et al., 1974; Thomas et al., 1974; Gilbert et al., 1975), conversion experiments (Carmody & Rattazzi, 1974; Beutler et al., 1975) and direct chemical analysis, a molecular model was proposed for hexosaminidases A and B (Geiger & Arnon, 1976; Lee & Yoshida, 1976; Beu+ From the Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel. Receioed October 26, 1977. its apparent molecular weight which is around 150 000 compared with 100 000 of the A and B isozymes. Immunochemical analysis indicates that the P isozyme is antigenically crossreactive with both A and B isozymes, but it does not contain the A-specific antigenic determinants, and exhibits identical antigenic specificity to hexosaminidase B. Two possible structures are suggested that are compatible with the experimental data: (a) a hexosaminidase B like structure with higher extent of glycosylation; (b) a hexamer of P chain, possibly arranged as three P 2 subunits. tler et al., 1976). According to this model, both hexosaminidases are built of two subunits, each subunit composed of two S-S-linked identical polypeptide chains. However, whereas hexosaminidase B is composed of four identical chains (P2,&), the A isozyme has one 8 2 and one cy2 subunit ( c y 2 P 2 ) (Geiger & Arnon, 1976). In the present study we have investigated some of the biochemical and immunochemical properties of hexosaminidase P, in an attempt to describe its structure in molecular terms. Materials and Methods Hexosaminidase A and B were purified from human placentae to an apparent homogeneity by the procedure described previously (Geiger et al., 1975; Geiger & Arnon, 1976). The purity of the preparations was established by analytical ultracentrifugation, electrophoresis on polyacrylamide gel in the presence of NaDodS04,' as well as by gel electrophoresis and isoelectric focusing under nondenaturing conditions. Sera of Pregnant Women. Serum samples were collected from women in the third trimester of pregnancy and 48 h after delivery. Sera used for estimation of enzymatic activity were not pooled and were stored at -20 "C until tested. Sera used for purification of hexosaminidase P were collected, pooled, and stored until used a t -20 "C. Hexosaminidase Assay. Enzyme solution (1 00 pL, diluted in 0.04 M citrate buffer, pH 4.4) was incubated for 10 min a t 37 O C with substrate (200 pL) containing 0.1 mg/mL 4methylumbelliferyl-N-acetyl-P-D-glucosaminide (Pierce) and 1 mg/mL bovine serum albumin (Grade A, Calbiochem). The Abbreviations used: NaDodS04, sodium dodecyl sulfate; PBS, phosphate-buffered saline; IEF, isolectric focusing. 0006-2960/78/0417-1713$01.00/0