Tohoku J. exp. Med., 1978, 125, 115-120

Fragmentation of Human Albumin with Proteolytic Enzymes and Its Antigenicity with Special Reference to Human-Specificity

KAORUSAGISAKA, YOKO SUGIYAMAand NOBORUTSUGAWA Department of Legal Medicine, Gifu University School of Medicine, Gifu 500

SAGISAKA, K., SUGIYAMA, Y. and TSUGAWA, N. Fragmentation of Human Albumin with Proteolytic Enzymes and Its Antigenicity with Special Reference to Human-Specificity. Tohoku J. exp. Med., 1978, 125 (2), 115-120 Human albumin prepared by the trichloroacetic acid method was treated with bromelin, ficin, papain, pronase or trypsin. Pronase or trypsin-treated albumin showed four fragments on polyacrylamide disc electrophoresis. When rabbit antiserum to

pronase-treated albumin was adsorbed with monkey albumin, antibody with human specificity was completely abolished, whereas anti-albumin serum adsorbed with pronase-treated albumin retained the antibody to human albumin. It was considered that antigenic site with human specificity in albumin structure was inactivated by pronase.„Ÿalbumin fragmentation; human-specificity

The species identification of blood stains found on the scene has been one of the most important problem in forensic science. Nowadays, it has been most solved by immunoassay with anti-human hemoglobin. On the other hand, human specificity of blood proteins other than hemoglobin has been investigated to determine species-specificity, indicating that human alpha 2-macroglobulin and albumin can be distinguished serologically from those of the other mammals (Tsugawa et al. 1971; Hara 1974; Bauer 1974). Recently, complete amino acid sequence of human albumin and antigenicity of trypsin fragments of bovine albumin have been studied (Habeeb et al. 1974; Behrens et al. 1975; Meloun et al. 1975; Atassi et al. 1976). Akiyama et al. (1977) investigated human specificity of cyanogen bromide or pepsin fragments of albumin. This paper is concerned with electrophoretic pattern of albumin fragments developed with proteolytic enzymes and human specificity of pronase fragments.

MATERIALS AND METHODS

Albumin. Albumin was prepared from serum by the method described by Schwert (1957). Pooled serum was mixed with one-tenth volume of 10% trichloroacetic acid. The resultant precipitate was separated by centrifugation at 10,000 rpm for 20 min. An equal volume of ethanol to the starting serum was added to the precipitate. After stirring with a spatula, the mixture was centrifuged at 10,000 rpm for 20 min. The supernatant was removed and dialyzed against tap water and then against saline. Albumin was

Received for publication, October 12, 1977. 115 116 K. Sagisaka et al.

lyophilized and stored at -20•Ž.

Treatment of albumin with proteolytic enzymes. Proteolytic enzymes, bromelin (Nutri tional Biochem. Co., U.S.A.), ficin (Wako Pure Chemicals, Tokyo), papain (E. Merk, Germany), trypsin (type 2000E/g, E. Merk, Germany) and pronase (type p, Kakenkagaku Co., Tokyo) were dissolved in the buffers described in the preceding paper (Sagisaka 1975). The mixture of the enzyme solution and albumin was incubated at 37•Ž for 1 hr and the digestion was stopped by heating at 70•Ž for 1 min. The treated albumin solution was freshly prepared at each experiment.

Preparation of rabbit antisera to albumins. Rabbits were intramuscularly immunized with 1% albumin and an equal volume of Freund's complete adjuvant. After 5 to 7 weekly immunizations the rabbits were bled. Antiserum to pronase-treated albumin

(p-albumin) was similarly prepared.

Polyacrylamide disc electrophoresis, agarose immunoelectrophoresis and double im

munodiffusion. Seven percent polyacrylamide gel of pH 8.9 and spacer gel of pH 6 .7 as described by Davis (1964) were used. After electrophoresis, the gels were stained with amidoblack 10 B. One percent agarose gel with veronal buffer containing calcium

lactate described by Hirshfeld (1960) was used. For double immunodiffusion, wells of 0 .5 cm in diameter and 0.25 cm of distance were cut on 1% agar gel in saline.

Isolation of pronase fragments. After treatment with pronase, p-albumin was dialyzed against 0.5 M carbonate buffer, pH 9.1. Then, p-albumin was conjugated with fluorescein isothiocyanate (FITC) at the ratio of 1/50 (FITC/ protein). The FITC conjugated

fragments were clearly detected in polyacrylamide gel under ultra-violet ray . Four fragments in the gel were cut off and homogenized in Potter's homogenizers. The in dividual homogenized gels were centrifugated at 10,000 rpm for 20 min and the supernatants were separated.

Adsorption of antiserum. Anti-albumin or anti-p-albumin serum was adsorbed with native human or monkey (Macaca f. fuscata) albumin or p-albumin (each 5 mg/ml) by mixing at the ratio of 1:1 in volume at room temperature for 2 hr. After centrifugation at 10,000 rpm for 20 min, the supernatant was taken.

Ring test. Glass tubes, 3 cm in length and 0.2 cm in diameter, were used .

RESULTS

Electrophoretic patterns of albumin treated with proteolytic enzymes . Albumin (2.5 mg/ml) was treated with a half volume of 0.5% bromelin, 0.1% ficin, 0.1% papain, 0.02% pronase or 0.1% trypsin at 37•Ž for 1 hr. After heating at 70•Ž for 1 min, the mixtures were subjected to polyacrylamide gel disc electrophoresis . As shown in Fig. 1, albumin prepared by the trichloroacetic acid method contained a very small amount of alpha-globulin which was estimated to be transferrin . The treatment of albumin with the enzymes resulted in fragmentation of albumin into two to several bands; especially treatment with pronase and trypsin were effective. By pronase treatment , four distinct bands each of which had relatively similar density were produced. The fragments produced by the treatments with bromelin, ficin and papain were faint and of fast mobility .

Influence of enzyme concentration on the pattern of fragmentation . Detailed study of proteolytic fragmentation was made with pronase only . A serial two-fold dilution of pronase solution (2 mg/ml) was prepared . To each dilutant twice volumes of albumin solution (8 .2 mg/ml) were added , and the mixture was in Fragmentation of Human Albumin 117

Fig. 1. Polyacrylamide gel disc electrophoretic patterns of albumin treated with various proteolytic enzymes. Track 1, native albumin; 2, trypsin-; 3, papain-; 4, ficin-; 5, bromelin-; 6, pronase-treated albumin. Note clear fragmentation of pronase-treated albumin.

Fig. 2. Polyacrylamide gel disc electrophoretic patterns of albumin treated with pronase of various concentrations. Track 1, native albumin; 2, albumin treated with pronase at a albumin/pronase ratio of 128:1; 3, 64:1; 4, 32:1; 5, 16:1; 6, 8:1. At lower concentrations of pronase, a fastly migrating faint fragment developed. Complete fragmentation took place at the ratio of 16:1 or more.

cubated at 37•Ž for 1 hr. When the ratio of albumin/pronase was lower than 32:1, faint fragments with fast mobility were produced. When the ratio was higher than 16: 1, distinct fragmentation with four bands was observed (Fig. 2). When incubated for 2 hr or more, clear pattern of fragments was no longer observed on disc electrophoresis.

Immunoreactivity of pronase fragments. The concentration of each fragment isolated from polyacrylamide gel was adjusted to 5 mg/ml, and its doubling dilutions were made up. Immunoreactivity of each fragment against anti albumin, which could react with albumin of up to 0.245 ƒÊg/ml, was investigated.

The minimum concentrations of the fragments 1, 2 and 3, which were designated from their mobilities, were 1.25, 0.65 and 0.65 ƒÊg/ml, respectively. As for the 118 K. Sagisaka et al.

Fig. 3. Immunoelectrophoretic patterns of native and p-albumin. Wells 1 and 3, native albumin; well 2, p-albumin, trough A, anti-p-albumim trough B, anti-albumin. P albumin produced a fine precipitation line (indicated by arrows) moving fastly which showed partial fusion with a main precipitation line. In cathodal area, p-albumin produced a precipitation (indicated by an arrow) with anti-p-albumin which was probably due to pronase. fragment 4, even undiluted solution (5 mg/ml) showed no reaction. Specificity of those fragments was investigated by double im munodiffusion method, showing that precipitation lines developed by the fragments 1, 2 and 3 fused completely with the line of native albumin. The fragment 4 did not produce any clear line. As shown in Fig. 3, p-albumin produced three precipitation lines with anti-p-albumin on immunoelectrophoresis, two of which were due to albumin derivatives and the other developed in cathodal area was caused probably by pronase. The two pre cipitation lines due to albumin derivatives showed partial fusion. This pattern was also observed between p-albumin and anti-albumin except the line in cathodal area. Antigenicity of p-albumin. The titers of anti-albumin and anti-p-albumin against human and monkey albumins are shown in Tables 1 and 2. As anti-p albumin had similar precipitin activity to anti-albumin, it was suggested that pronase treatment little affected the antigenicity of albumin. However, when those antisera were adsorbed with monkey albumin, different precipitin activity was observed. Anti-albumin adsorbed with monkey albumin maintained yet potent

TABLE1. Precipitin activityof anti-albuminin the ring test beforeand after adsorptionwith human, monkeyor p-albumin

* native human albumin Fragmentation of Human Albumin 119

TABLE2. Preciptin activity of anti-p-albumin in ring test before and after adsorption with human, monkey or p-albumin

precipitin activity specific for human albumin. On the other hand, in anti-p albumin adsorbed with monkey albumin, antibodies not only to monkey albumin but also to human one disappeared completely (Table 2). Immunoreactivities of human albumin and p-albumin were compared by adsorption of anti-albumin.

Human albumin abolished completely antibodies to human and p-albumin. On the contrary, p-albumin adsorbed scarcely the antibody to human albumin.

DISCUSSION

The species determination of blood stains has been performed serologically

with anti-human hemoglobin. For examination of stains of body fluids such as

saliva, sweat and semen, immunoassay with anti-human serum or fibrin plate method is adopted. Recently, human specificity of serum proteins has been

investigated. Bauer (1974) reported that human albumin consisted of 6 antigen

determinants, two of which were shared by albumins of primate animals and the

others were common to mammalian one. On the other hand, many proteolytic

enzymes have been applied to elucidate the structure of albumin. Chymotrypsin and pepsin treatments of human albumin produced two to three fragments, of which

reactivities were proved to differ from native albumin on immunoelectrophoresis

(Lapresle et al. 1959). Pronase and trypsin splitted albumin into three to four fragments, but the other enzymes produced few definite fragmentation under the

condition of this experiment. When albumin was treated at an albumin/pronase

ratio of 8:1 (w/w) at 37•Ž for 1 hr, four fragments could be clearly demonstrable.

Pronase, which was extracted from streptomyces griseus, is known to have the

broadest specificity as proteolytic enzyme. The present result indicates that several points of albumin structure were sensitive to pronase. The study of

immunoreactivity of p-albumin with anti-albumin revealed that pronase reduced

evidently the reactivity of albumin. Richard et al. (1960) reported also that

bovine albumin treated with chymotrypsin showed extremely low reactivity. Two

precipitation lines of p-albumin were observed on immunoelectrophoresis. But the result of the double immunodiffuusion revealed that immunogenicity of the

fragments was identical with that of native albumin. Judging from the titration of

crude anti-p-albumin, it appeared that antigenicity of albumin was not affected by

pronase treatment. However, the adsorption experiment revealed that anti-p albumin did not contain the antibody with human specificity. In addition, the 120 K. Sagisaka et al.

antibody was retained still in anti-albumin adsorbed with p-albumin. These results suggest that some sequence(s) bearing the human specificity in albumin structure might have been disrupted by pronase treatment. Akiyama et al. (1977) investigated species-specificity of cyanogen bromide fragments of human albumin, and pointed out that the fragment containing C-terminal sequence inhibited the antibody activity to both human and monkey. Moreover, the author revealed that antiserum to the fragment had no antibody with human specificity. From the results of reactivity of p-albumin and property of anti-p-albumin, it may be concluded that antigenic site with human specificity in albumin structure is inactivated by pronase.

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