Acidic Protease from Human Seminal Plasma PURIFICATION AND SOME PROPERTIES OF ACTIVE ENZYME AND OF PROENZYME*

A procedure to purify to homogeneity the active form as well as the proenzyme form of the acidic protease of human seminal plasma is described. This involved precipitation with ammonium sulfate, chromatography on diethylaminoethylcellulose, Sephadex G-200, and Sephadex G-100. The molecular weights of the active form and of the proenzyme were determined by electrophoresis and gel filtration to be 35,000 and 42,000, respectively. The proenzyme was more stable than the active form in alkaline solution and can be converted into the active enzyme under acidic conditions. The active form of the acidic protease can hydrolyze hemoglobin, N,N’-dimethylcasein, N-acetyl-L-phenylalanyl-L.-diiodotyro-sine, and N-benzyloxycarbonyl-L-glutamyl-L-phenylalanine, but cannot hydrolyze bovine serum albumin, ovalbumin, N-benzyloxycarbonyl-L-glutamyl-L-tyrosine. The active form was also inhibited by p-bromophenacyl bromide and 1,2-epoxy-3-(p-nitrophenoxy)propane.

Although several proteases are known to exist in the acrosome of mammalian sperm and in seminal plasma, few have been purified and characterized.
A trypsin-like enzyme (acrosin) has been purified from the acrosomes of boar as well as human sperm (l-4). An acidic protease with a pH optimum of 2.8 has been partially purified from the acrosomes of bull and ram sperm (5). A neutral protease with a pH optimum of 8.0 has been purified 25-fold from human seminal plasma (6). An acidic protease in human seminal plasma was originally described as pepsin and its proenzyme as pepsinogen (7). The active form of this enzyme has recently been partially purified and characterized (8). The functions of these proteases are not yet well understood.
Acrosin seems to be necessary for the removal of the zona pellucida of an ovum prior to the process of fertilization (9). The neutral protease in seminal plasma has been shown to be able to digest proteins in seminal plasma and cervical mucus and may thereby facilitate the migration of sperm in these fluids (10). The function of the acidic proteases in the acrosome and in seminal plasma has not been demonstrated.
The present paper will describe a method for the purification to homogeneity of the acidic protease and its proenzyme from human seminal plasma. Some of their properties will also be presented.
The activity of - substrates by the method of Kassell and Meitner (ll), and with synthetic substrates by the method of Ryles (12).

Determination of Protein
Protein concentration was determined by the method of Lowry et al. (13) using bovine serum albumin as the protein standard. During column chromatography, protein content in each fraction was determined by measuring the absorbance at 280 nm.

Purification of Active Form of Acidic Protease
Pooled fresh human semen was centrifuged at 1,000 x g for 10 min at 4' to remove spermatozoa. The cell-free seminal plasma was kept at -20' until use. When a sufficient amount of the seminal plasma had accumulated, it was thawed and clarified by centrifugation at 20,000 x g for 20 min at 4'. Unless stated otherwise, the following steps were carried out at 4'.
Step 1: Ammonium Sulfate Fractionation-The clear seminal plasma was brought to 40% saturation of ammonium sulfate by the slow addition of the solid salt. After standing for 1 hour, the precipitate was removed by centrifugation at 20,000 x g for 15 min. The protease activity was then precipitated at 70% saturation of ammonium sulfate. The precipitate was collected by centrifugation and redissolved in 0.02 M sodium phosphate buffer, pH 6.5.
Step 2: Column Chromatography on DEAE-cellulose-A concentrated solution of NaCl was added to the protein solution until the final concentration of the salt was 0.2 M. The solution was then applied onto a DEAE-cellulose column (40 x 3.5 cm) which was pre-equilibrated with 0.02 M sodium phosphate buffer, pH 6.5, containing 0.2 M NaCl. A large bulk of protein including a neutral protease was not retained and washed out of the column with the buffer. Then a linear gradient of NaCl was applied to the column. The acidic protease activity was eluted between 0.3 and 0.4 M NaCl. The fractions containing the activity were pooled and concentrated by using Aquacide. A typical chromatographic profile is shown in Fig. 1.
Step 3: Column Chromatography on Sephaden G-200-The concentrated solution was adjusted to pH 3.0 by the addition of 2 M HCl and incubated at 37" for 1 hour. The solution was then applied onto a column of Sephadex G-200 (50 x 2.5 cm) which was pre-equilibrated with 1 mM HCl containing 0.1 M NaCl. The fractions containing the acidic protease were then pooled and concentrated.
Step 4: Column Chromatography on Sephadex G-ZOO-The concentrated solution was further chromatographed on a column of Sephadex G-100 (80 x 1.2 cm) pre-equilibrated with 1 mM HCl containing 0.1 M NaCl. A typical chromatographic profile is shown in Fig. 2. The fractions containing the enzyme activity were pooled, dialyzed against double distilled water, and lyophilized. The protease could then be kept at -20" for several months without appreciable loss of the enzyme activity.
A summary of a representative purification is shown in Table I.

Purification of Proenzyme of Acidic Protease
The proenzyme of the acidic protease could be purified by the procedure described above with the following alterations. The protein solution with the activity of the acidic protease from Step 2 was not acidified. The gel filtrations (Steps 3 and 4) were carried out with the columns pre-equilibrated with 0.02 M sodium phosphate buffer, pH 6.5. The fractions containing the proenzyme could be identified by the assay of Kassell and Meitner (11) because the proenzyme is converted into the active form under the acidic conditions of the assay. The chromatographic profiles of the proenzyme were similar to those of the active enzyme. The result of the purification of the proenzyme was similar to that of the active enzyme shown in Table I. FRACTION NUMBER FIG. 1. Chromatographic profile of ammonium sulfate fraction on a DEAE-cellulose column (40 x 3.5 cm). The column was equilibrated with 0.02 M sodium phosphate buffer, pH 6.5, containing 0.2 M NaCl. A linear gradient of NaCl from 0.2 to 0.9 M in the same buffer was applied when washing was completed (indicated by the arrow). Protein concentration was determined by absorbance at 280 nm, proteolytic activity by the method of Kassell and Meitner (11) with hemoglobin as substrate, and NaCl concentration by conductivity measurement. Fractions 35 to 70 were diluted lo-fold with buffer prior to the determination of absorbance at 280 nm. Step 3 on Sephadex G-100 (80 x 1.2 cm). The column was pre-equilibrated and eluted with 1 mM HCl, pH 3, containing 0.1 M NaCl. Protein concentrations and proteolytic activity were determined as indicated in Fig. 1. 7575 Electrophoresis Estimation of the molecular weight by polyacrylamide gel electrophoresis was done in the presence of Na dodecylS0, according to the method of Weber and Osborn (14).

Homogeneity, Molecular
Weight, and Stability-The active form and the proenzyme of the acidic protease of human seminal plasma were purified to homogeneity by the procedures described above. They appeared as single bands in polyacrylamide gel electrophoresis in the presence of Na dodecyl-SO, (Fig. 3). The active form moved slightly faster than the proenzyme. The appearance of the purified proenzyme as a single band in the gel rules out the possibility of any peptide inhibitor noncovalently bound to the enzyme in the preparation. The homogeneity was also confirmed by polyacrylamide gel electrophoresis in 7% gel with Tris-HCl buffer, pH 6.6, performed according to the method of Davis (15). Mobilities of the active form and of the proenzyme in the polyacrylamide gel electrophoresis in the presence of Na dodecyl-SO, were used to estimate their molecular weights. From the standard curve (Fig. 4), the molecular weights of the The activity of the acidic protease was assayed with acid-denatured hemoglobin as substrate (11). One unit is the amount of the enzyme required to cause an increase of 1 absorbance unit at 280 nm in 30 min. Protein was quantitated by the method of Lowry et al. (13) 3. Polyacrylamide gel electrophoresis in 0.1% Na dodecyl-SO, of (a) proenzyme, (b) active acidic protease, and (c) proenzyme converted into active enzyme. Samples were preincubated in 0.01 M sodium phosphate buffer, pH 7.0, containing 0.1% Na dodecyl-SO, and 0.1% @-mercaptoethanol at 37" for 3 hours before electrophoresis which was performed according to the method of Weber and Osborn (14). BSA, bovine serum albumin. active form and of the proenzyme were estimated to be 35,000 and 42,000, respectively. These values were confirmed by gel filtration using Sephadex G-100 chromatography. They were also quite close to those of hog pepsin and of pepsinogen (12).
pH Stability-The active form of the acidic protease was quite stable between pH 2 and 6.5. Above pH 6.5 its activity dropped sharply (Fig. 5). The proenzyme was quite stable between pH 6 and 9. Above pH 9, it was quickly denatured. Below pH 6, the proenzyme was converted into the active form of the acidic protease. The data supporting the conversion is given below.
The pH dependence of the activity of the purified acidic protease was similar to that of a partially purified preparation (8) but the pH optimum was slightly shifted from 3.0 to 2.5.
Conversion of Proenzyme into Active Form--When the preparation of the proenzyme was adjusted to pH 3 with 2 M HCl and then incubated at 37" for 1 hour, some small peptides were released. This resulted in an extra peak of a smaller molecular weight material in the elution profile of a Sephadex G-50 column (Fig. 6). Moreover, the acid-treated proenzyme showed a slight reduction in its mobility in the column of Sephadex G-50 (Fig. 6). It also possessed activity and electrophoretic mobility identical with those of the purified active form (Fig. 3). Thus, the proenzyme was converted into its active form at pH 3. Our additional data, not shown here, indicated that acidic conditions (pH 2 to 5) would initiate the conversion.
Activities of Active Form-The purified acidic protease can hydrolyze hemoglobin, N,N'-dimethylcasein, but neither ovalbumin nor bovine serum albumin (Fig. 7). The apparent K, values for hemoglobin and N,N'-dimethylcasein were 1.25 and 1.8 mg per ml, respectively. It was noted that a high concentration of N,N'-dimethylcasein was inhibitory to the enzyme. This effect was similar to that observed with trypsin (16). The mechanism of inhibition is unknown. As shown in Table II, the specific activity of the purified acidic protease was quite comparable to that of commercial hog pepsin, when hemoglobin was used as substrate. The sensitivity to inhibition by 1,2-epoxy-3-(p-nitrophenoxy)propane and by p-bromophenacyl bromide was also similar to that of hog pepsin. However, the acidic protease was loo-fold less active than hog pepsin when N-acetyl-L-phenylalanyl-L-diiodotyrosine was used as substrate. The protease could also hydrolyze N-benzyloxycarbonyl-r,-glutamyl-L-phenylalanine but not N-benzyloxycarbonyl-L-glutamyl-L-tyrosine.  buffer, pH 6.5. Bottom, proenzyme was incubated in 1 mM HCl at 37" for 1 hour and then chromatographed in 1 mM HCl, pH 3. DISCUSSION The purification procedure described in this paper differs from that reported earlier (8) mainly in the use of DEAE-cellulose as an ion exchanger instead of CM-cellulose. This change allowed a larger amount of nonenzyme protein to be washed out by the buffer (Fig. l), resulting in a better purification.
In fact, column chromatography on DEAE-cel-  lulose was the best step in the purification procedure, giving the largest increase in the specific activity of the enzyme ( Table I). The present procedure is far better than the previous one (8) in that it gives a homogeneous preparation of enzyme, but the final yield is rather low. The major loss of activity seems to be at the first two steps. This may be due to the action of the neutral protease in seminal plasma (6) which is not removed until the second step.
The present procedure can be used to purify the active form of the acidic protease as well as its proenzyme. When all steps of the purification were carried out at pH 6.5, only the proenzyme form was obtained.
But when the gel filtrations (Steps 3 and 4) were carried out at pH 3 only the active form was obtained. Since the yield and specific activity of the active form were identical with those of the proenzyme at each step of the purification, it was likely that the proenzyme was the only form present in human seminal plasma at physiological pH of 7.0 to 7.5 (17). This was fully supported by the stability of the proenzyme and the instability of the active form at pH 7.5 (Fig.  5).
The following data suggested that the preparation obtained 7577 complex between the active enzyme and a protease inhibitor. (a) The preparation at pH 6.5 could be converted into the active enzyme in acidic conditions (pH below 6, Fig. 5). This was similar to the acid-dependent conversion of pepsinogen into pepsin (12). The conversion did not take place at high concentrations of sodium chloride during chromatography on DEAE-cellulose.
If an enzyme-inhibitor complex was present,