STUDIES OF THE HEMAGGLUTININ OF HAEMOPHILUS PERTUSSIS HIDEO FUKUMI, HISASHI SHIMAZAKI, SADAO KOBAYASHI AND TATSUJI UCHIDA

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1 STUDIES OF THE HEMAGGLUTININ OF HAEMOPHILUS PERTUSSIS HIDEO FUKUMI, HISASHI SHIMAZAKI, SADAO KOBAYASHI AND TATSUJI UCHIDA The National Institute of Health, Tokyo, Japan (Received: August 3rd, 1953) INTRODUCTION It is a well-known fact that Haemophilus pertussis has a substance capable of agglutinating red blood cells, but the chemical characteristics, or the biological significance of this substance, called hemagglutinin, have not been much investigated. As far as we know, Masry's paper(1) is the only one, which dealt with this substance in a relatively pure state. According to Masry(1), Ungar(2) or Fisher(3), the substance hemagglutinin is inactivated at 60 Ž. Masry(1) obtained partially purified hemagglutinin by precipitation with cold methanol 40% and elution in distilled water. In these respects, the hemagglutinin is presumed to be of protein nature. On the other hand, Yamamoto, Zenyoji, and Kato(4) obtained a substance from Haemophilus pertussis which showed hemagglutination reaction in high dilution, and they stated that the substance is of carbohydrate nature. We think however that the substance obtained by them is not the one which really shows hemagglutination reaction in Haemophilus pertussis suspension. Attempts were made by our laboratory to extract and purify the hemagglutinin of Haemophilus pertussis and some experiments were made to elucidate its characteristics by using partially purified material. This publication presents some of the findings obtained by us so far. THE METHOD FOR TITRATION OF THE HEMAGGLUTININ As for the method for titration of the hemagglutinin, each worker has employed his own and it may be said that there has been no standard method. Ungar employed 5% of red blood cell suspension and read the reaction, shaking after incubation in a water bath at 46 Ž for two hours, but we do not think his method is to be employed. Masry read the reaction according to the pattern of the sedimented cells, following Keogh, and North(5). The concentration of red blood cell suspensions has been also variable for each worker. For instance, Masry employed 1%, while 5% was used by Thiele(6). Most workers employed 37 Ž for the reaction temperature, but according to Masry, the results were not varied in the temperature range 4-50 Ž. The method for hemagglutinin titration employed by the present authors is as follows: 641

2 642 FUKUMI et al To 0.5 cc of serial two-fold dilutions of hemagglutinin solution, 0.5 cc of 0.25% suspension of red blood cells was added. After about 3 hours at room temperature, the end point was read according to the patterns of the sedimented cells. Physiological saline solution was always employed for the diluent. According to our preliminary experiment, the end point value was not much different whether chicken or sheep red blood cells were employed. Therefore, chicken red blood cells were mostly used in the experiments below. STABILITY TEST Preliminary to the extraction and purification experiments, the stability of the hemagglutinin to several chemical compounds was investigated. When cell suspension of Haemophilus pertussis was exposed to 10% solution of K2CrO4 at room temperature for one hour, its hemagglutination titer was reduced to one half or one fourth. The exposure to ethylalcohol or aceton for one hour destroyed almost entirely the hemagglutinating capacity of bacterial cell suspension. 4% of boric acid also reduced the hemagglutinating activity of cell suspension to about one half or one fourth for one hour at room temperature. As for methanol, experiments with bacterial cell suspension were not carried out yet, but we recognized the complete loss of activity in partially purified preparation (see below), after precipitation with methanol at room temperature. In this respect, the hemagglutinin which we are now dealing with, is considered to be a different substance from the substance of carbohydrate nature prepared by Yamamoto et al (4) having the capacity to hemagglutinate. In the above experiments, it has been clear that aceton, alcohol or methanol could not be used for extraction or purification of the hemagglutinin at least at room temperature. EXTRACTION Masry(1) examined serveral methods for extraction of the hemagglutinin and reported that he obtained good results with 2M of NaCl solution or 1M of sodium acetate solution but is pessimistic for freezing-and-thawing treatment. Our experimental results with freezing-and-thawing method was as follows: 48 hours' growth of Haemophilus pertussis (Strain 10536) *on Bordet-Gengue medium was collected and washed with saline by ordinary centrifugation. The washed bacterial bodies were suspended in a little amount of saline and freezthawn five times. A little more amount of saline was added and centrifuged down. The supernatant was called as S1. The precipitate was again suspended in a little amount of saline and f reez-thawn five times. It was added with a * The strain was employed in all of the experiments reported in this publication.

3 PERTUSSIS 643 little more amount of saline and centrifuged down. The supernatant was named as S2. The sediment was resuspended with the original amount of saline. This suspension was called as P1. Table 1 The hemagglutinating capacity of S1, S2, P1, and the original suspension was summarized in Table 1. This indicates that the hemagglutinin was not fully extracted with the ten times freezing-thawing as in the above experiments. P1 was then again five times f reez-thawn and centrifuged down. The supernatant was named as S3. The sediment was resuspended with the original amount of saline. This was named as P2. The hemagglutinating capacity of S3 and P2 were shown in Table 2. Table 2 From this experiment, it was concluded that the freezing-and-thawing treatment could extract the hemagglutinin almost fully from bacterial bodies if repeated at least 20 times. In the following experiments, the extraction of the hemagglutinin from H. pertussis was exclusively made with the freezing-and-thawing treatment repeated 30 times. SALTING OUT As the hemagglutinin was thought to be of protein nature according to its heat-sensitiveness and other characteristics reported by some workers, it was attempted to purify this substance by salting out with ammonium sulfate. In a preliminary experiment we found that the hemagglutinin was almost entirely precipitated from a f reez-thawn extract of H. pertussis by addition of ammonium sulfate to half saturation, and further a considerable part of the hemagglutinin

4 644 FUKUMI et al was precipitated from the saline solution of the above precipitate with ammonium sulfate of one fourth saturation. We conducted then further experiments as below. A f reez-thawn extract of H. pertussis was added with ammonium sulfate to one fourth saturation. The precipitate was resuspended with the original amount of saline (1/4P). The supernatant was further added with ammonium sulfate up to one half saturation and the resulting precipitate was resuspended with the original volume of saline (1/2P). The supernatant was dialyzed against physiological saline and then concentrated to the original volume by evaporation through dialyzation sack (1/2S). The hemagglutinating capacity of these materials is shown in Table 3. Table 3 The table shows that 1/4P was rather a little higher in its hemagglutinating ability than 1/2P. According to our repeated experiences, the hemagglutinating abilities of 1/4P and of 1/2P were mostly about equal. In the above experiment, the solution 1/4P was much clearer than the 1/2P and from this it may be presumed that 1/4P was much purified for the hemagglutinin than 1/2P. The solution 1/2P was again precipitated with ammonium sulfate in the final concentration of one fourth saturation and half saturation and the resolution of either 1/4P or 1/2P was called 1/4P-2 or 1/2P-2 respectively. The hemagglutinating ability was as shown in Table 4. Table 4 From the above experiments we considered that the hemagglutinin can be partially purified by salting-out with ammonium sulfate of half saturation. while purer preparations may be obtained by ammonium sulfate of one fourth saturation, the considerable loss of the hemagglutinin is expected to be inevitable. In the following experiments partially purified hemagglutinin solutions

5 PERTUSSIS 645 by means of half saturation ammonium sulfate after freez-thawing extraction were employed throughout.* ABSORPTION OF THE HEMAGGLUTININ WITH RED BLOOD CELLS It is well known that there were several kinds of viruses capable of agglutinating red blood cells of certain animals. These viruses were divided into two groups. The members of the one group have a receptor destroying enzyme, therefore elute from the red blood cells after a certain incubation period, which have absorbed them, while the viruses of the other group have no such enzymatic activity (7). The fact that the hemagglutination with the hemagglutinin of H. pertussis is due to the absorption of the substance on to red blood cells is very clear. If hemagglutinin was mixed with red blood cells and let stand for a certain time at room temperature, the supernatant fluid becomes deprived of hemagglutinin. However, if the sedimented red blood cells were resuspended in phosphate buffer and let stand about two hours at 37 Ž, no elution of hemagglutinin was recognized at all in the supernatant. This experiment indicates that the hemagglutination of H. pertussis is not of the same category with that of influenza virus whose hemagglutination is accompanied with destruction of the receptor substance on the surface of red blood cells. ph STABILITY OF THE HEMAGGLUTININ OF H. Pertussis The ph stability is a very important characteristic either for obtaining some inf ormations of the essential properties of a certain substance or for establishing a purification method of it, therefore we attempted first to know the ph stability of the hemagglutinin of H. pert ussis. As the hemagglutinin, we employed partially purified preparation obtained by half saturation of ammonium sulfate after freez-thawing. Citrate buffer solutions, phosphate buffer solutions or glycine buffer solutions were used for test medium according to the ph ranges required. The exposure to a given ph of the hemagglutinin was conducted in the way that hemagglutinin solution in a given ph was incubated at 37 Ž for one hour, then neutralized by acid or alkali and the hemagglutinating capacity was measured. The experimental results are summarized in Fig. 1, which shows that the hemagglutinin is fairly stable in the ph range of 3-9. We recognized in this * The experiments concerning salting out with ammonium sulfate were mainly conducted in the winter time in which the room temperature was kept at about Ž. When we followed the same procedure to obtain a partially purified preparation of hemagglutinin in warmer seasons, we found that salting out was not so fruitful as before. After some additional experiments, we thereafter carried out this procedure successfully at the temperature 5-10 Ž using a refrigerated centrifuge.

6 646 FUKUMI et at experiment that precipitation was formed below about ph 5, so when measuring the hemagglutinating activity, we did it after enough shaking the neutralized test materials. However, we next attempted to know which of precipitate or supernatant possesses the hemagglutinating ability. The hemagglutinin solution was adjusted to ph 4.5 by addition of citrate buffer, and the resulting precipitate was separated from the supernatant fluid by centrifugation. The supernatant and the precipitate resuspended in phosphate buffer of ph 7.4 were separately tested for their hemagglutinating ability. The hemagglutinating ability proved to move completely to the precipitate. Fig. 1. ph stability of hemagglutinin of H. pertussis. We tried then to make use of acid precipitation of the hemagglutinin for its purification. A hemagglutinin solution partially purified by half saturation of ammonium sulfate was adjusted to ph 4.0 by addition of citrate buffer and the precipitate was again suspended in phosphate buffer of ph 7.4. We repeated this experiment several times but the yields never exceeded 50%. ACTION OF FORMALDEHYDE TO THE HEMAGGLUTININ The hemagglutinating activity of the influenza virus is well reserved even after formaldehyde inactivates its infectivity. Formaldehyde has no influence on hemagglutinating capacity of this virus particle even at 2mg/cc though a little reduction of activity is seen at 10mg/cc(8). In this experiment, we examined the influence of formaldehyde on the hemagglutinin of H. pertussis. The result of the experiment was summarized in Table 5. A hemagglutinin solution

7 PERTUSSIS 64 7 partially purified by half saturation of ammonium sulfate was employed as a test material. The measurement of formaldehyde was conducted according to the method using dimedon. The exposure to formaldehyde was carried out at 37 Ž for 30 minutes, and after that dialysis against physiological saline was made overnight before measurement of hemagglutinating activities. Table 5. Action of formaldehyde to the hemagglutinin As shown in Table 5, the hemagglutinin of H. pert ussis is completely inactivated by formaldehyde at a concentration of 2mg/cc. The exposure to formaldehyde was conducted in buffer solutions so that ph of the reaction mixtures was kept at Therefore it was not acid reaction but formaldehyde itself that inactivated the hemagglutinating activity. At first we attempted to investigate influence of formaldehyde upon the hemagglutinin in order to know whether formaldehyde be able to, fix the hemagglutinating capacity of the substance, because it might be useful to fix such a labile substance as the hemagglutinin of H. pertussis in some way before the procedure of its purification. But it proved not to be able to apply this idea for this substance. DISCUSSION As Masry stated, the hemagglutinating substance of H. pertussis studied by Ungar might be a different type of hemagglutinin system. In his experiments, the hemagglutination took place at temperatures between Ž and the reading was made after resuspension of deposited red cells by shaking. The hemagglutinating substance purified from H. pertussis by Yamamoto et al, which is of carbohydrate nature, is also considered to be another type of substance. It is very much stable than the hemagglutinin studied by us and can be precipitated by methanol at room temperature with no deterioration. The hemagglutinin studied by us is considered to be the same substance studied by Masry, Fisher or Keogh and North. It is very unstable. It is inactivated at 60 Ž for a few minutes, or by aceton, alcohol, or methanol. It is largely precipitated by ammonium sulfate of half saturation, or in the ph

8 648 FUKUMI et al 4-5. It is more or less stable in the ph range It is deteriorated by formaldehyde at the concentration 2mg/cc or more. The hemagglutinin is absorbed on red blood cells, but is not eluted from them in the same way as influenza virus particles are leuted from red cells, which have absorbed them. SUMMARY 1. The hemagglutination of H. pertussis is extracted by freezing and thawing. 2. It is further purified by half saturation of ammonium sulfate. 3. It is precipitated at ph and resuspended at ph 7.4 with a little loss of activity. 4. It is inactivated at 2mg/cc or more of formaldehyde. REFERENCES 1. Masry, F. L. G.: Production, extraction and purification of the hemagglutinin of Haemophilus pertussis, J. gen. Microbiology, 7, , Ungar, J.: Agglutinationn of red blood corpuscles by H. pertussis, J. Path. & Bact., 47, , Fisher, S.: The haemagglutinin of Haemophilus pertussis. II. Observations on the structure of the haemagglutinating complex of culture supernatants, Australian J. exp. Biol. Med. Sci., 28, , Yamamoto, A., Zenyoji, H., and Kato, T.: Studies on H. pertussis. III. Extraction of the cell-components and its immunological property, Gumm.a J. Med. Sci., 1, , Keogh, E. V., and North, E. A.: The haemagglutinin of Haemophilus pertussis. I. Haemagglutinin as a protective antigen in experimental murine pertussis, Australian J. exp. Biol. Med. Sci., 26, , Thiele, E. H.: Studies on the hemagglutinin of Hemophilus pertussis, J. Imm., 65, , Hirst, G. K.: Hemagglutination as applied to the study of virus infection, in Delbrð¹ick, M.: Viruses 1950, California Institute of Technology, 44-51, Stanley, W. M.: The preparation and properties of influenza virus vaccines concentrated and purified by differential centrifugation, J. exp. Med., 81, , 1945.