The purification and preliminary investigation of fumarase, peroxidase, diamine oxidase and adenosine deaminase from human seminal plasma [proceedings].

The biochemistry of human semen is poorly understood, and despite initial studies by Mann (1964) little is known about the metabolism of spermatozoa1 cells, and even less about the seminal plasma. We have attempted the simultaneous purification of fumarase, peroxidase, diamine oxidase and adenosine deaminase from human seminal plasma, and compared the properties of these enzymes with those from other human and animal sources. Fumarase was determined by the method of Racker (1950), diamine oxidase by the method of Bardsley et al. (1 972), peroxidase by using 2,2’-azinodi-(3-ethylbenzthiazoline-6-sulphonic acid) as chromogen (Childs & Bardsley, 1975) and adenosine dearninase by following adenosine utilization at 270nm. The samples were prepared from pooled specimens of normal and oligospermic semen obtained from clinics at St. Mary’s Hospital, Manchester. Table 1 shows the simultaneous purification of the four enzymes, which were eluted consecutively from the first DEAE-Sephadex column. All were prepared to homogeneity or near-homogeneity as shown by polyacrylamide-gel electrophoresis. Adenosine deaminase from bay scallop (Aequipecten irradans concentricus) has been shown to obey Michaelis-Menten kinetics (Harbison & Fisher, 1973). Adenosine deaminase from seminal plasma resembled this enzyme in its kinetic behaviour. It has been suggested that purified seminal-plasma diamine oxidase has a similar substrate specificity to the human placental enzyme (Holtta et al., 1975). The eluate from concanavalin A-Sepharose 4B chromatography before application of a-methyl glucoside contained diamine oxidase, although the main peak was after elution with the glucoside. The initial elution of activity may represent enzyme with a lowered carbohydrate content relative to the glycoprotein eluted after a-methyl glucoside, although all active fractions oxidized [lysine]vasopressin, [argininelvasopressin and p-dimethylaminomethylbenzylamine, as judged by coupling the reaction to peroxidase with 2,2’-azinodi-(3-ethylbenzthiazolined-sulphonic acid) as chromogen. It therefore appears that the seminal-plasma diamine oxidase is similar to the human placental enzyme in its lysyl oxidase activity, rather than to the pig kidney enzyme, which does not oxidize these substrates (Crabbe et al., 1976). Peroxidase, purified from seminal plasma to homogeneity as shown by disc gel electrophoresis, exhibited similar spectral properties to horseradish peroxidase on addition of HzOz followed by ascorbic acid. The spectrum of active semen peroxidase had a marked shoulder at 407nm, which disappeared if the enzyme was left at room temperature for several days, the resulting protein being inactive. Haematin, with no peroxidase activity, was separated from horseradish apoprotein by a modification of the method of Teale (1959), and recombined with the inactive seminal-plasma apoprotein (Makino & Yamazaki, 1972) to yield a holoprotein with a marked shoulder in the spectrum at 400nm. This recombined peroxidase had a pH optimum at 4.4, whereas native seminalplasma enzyme had pH optimum 4.35, and horseradish peroxidase at pH4.1. The seminal-plasma enzyme also exhibited bactericidal properties similar to myeloperoxidase (Klebanoff, 1968). Fumarase was eluted from the Sephadex G-150 column as a major inactive protein band straddled by highly active protein. Addition of an extract of the central pooled protein peak to either of the pooled active fractions caused complete inhibition of activity. Polyacrylamide gels showed the presence of two major bands in both active