Purification and properties of a plant Agglutinin
Everhart, Donald Lee
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This study involved work with extracts of the seeds of Bauhinia purpurea alba, in which an N specific lectin was found by Mäkelä (37) and Boyd and McMaster (16). There were two main purposes in mind. First, to see if the lectin could be of practical use as a typing sera; and secondly, to get some insight into its chemical make-up. After working out a purification method and a method for checking the agglutinating power of the lectin, the lectin was tested against several small samples. The writer then tested this lectin against 90 random blood samples. The lectin disagreed with rabbit anti-N in one case -- the lectin typed one MN as an M. There is as yet no known reason for this difference, so it would seem that one could not use the lectin as a typing sera at the present time. The lectin was treated with formaldehyde and lost all its activity as a result. Erythrocytes were treated with an enzyme, ficin, and were then reacted with the lectin. The results showed an increase in the titer of the lectin, and the specificity of the lectin was lost. The freezing of a sample of the lectin for four months resulted in the loss of some of its activity. It remained specific, however. Upon dialyzing the crude extract against saline or water, the substances (Y) that remained behind in the membrane became nonspecific. This showed that there were at least two parts to the extract, one of large molecular weight because it would not pass through the membrane, and the other smaller in size because it would pass through the membrane. The substance that passed through the membrane was called X. By dialyzing the crude extract against distilled water and lyophilizing the distilled water, a substance was found which gave the reaction of a sugar and also seemed to have either or both glucosamine and galactosamine present. Since X showed the presence of reducing sugars and could be shown to inhibit Y against the M site on erythrocytes (see Table 7), it was thought that some sugars should be tested against Y to see if any of them would be able to inhibit Y. The same sugars were also tested against the lectin (Table 5). It was found that none of the sugars had any effect on the lectin; but raffinose, melibiose, and galactose all were found to inhibit Y (Table 8). It has also been shown by other workers (13) that lactose will inhibit Y. The inhibition of the sugars against lectin and Y were run in three ways. The first was the usual inhibition method where the inhibiting substance (sugar, in this case) is placed with the substance to be inhibited (Y) and incubated for one hour. Then the appropriate erythrocyte was added. The second method was to place erythrocytes, Y, and sugar all in the same tube at the same time. The third was to incubate sugar and erythrocytes together and then add Y. The results are given in Table 9. It was found by the writer that Method 2 was the best. This agrees with Krüpe as cited in Mäkelä (37) who states that if an appropriate sugar is added to erythrocytes that are agglutinated by a lectin, the lectin will leave the erythrocytes and pick up the sugar. This is shown by the breaking up of the clumps of erythrocytes. The sugars that would inhibit Y were tried against adsorbed rabbit sera, both anti-M and N. The sugars showed no effect against the rabbit sera. The writer feels that the lectin may not be as specific as the rabbit sera; thus, these inhibiting sugars may only be closely related to the true configuration of the M site.
Thesis (M.A.)--Boston University