17] ACTIVE FRAGMENT OF BOVINE SERUM ALBUMIN 291 



ratio of haptene to antigen of more than 1000/1) suggesting that the haptene 

 may be only a small part of the actual combining site. The only attempt to 

 isolate an antigenic combining site from a protein was carried out by Land- 

 steiner (1942). He used silk fibroin as antigen rendered soluble by treatment 

 with acid and increased its antigenic power by adsorption onto charcoal 

 before injecting it into rabbits. The silk fibroin was then degraded by strong 

 sulphuric acid and the digest was tested for its ability to inhibit the com- 

 bination of antigen and antiserum. Evidence was obtained showing that 

 apparently small peptides would inhibit this combination and that they 

 must retain therefore some of the features of the combining sites of the 

 original antigen. With the methods available at the time it was not possible 

 to characterize the active fragments further and as the original antigen was 

 partially degraded fibroin protein it was not clear what significance these 

 results bore relative to the nature of the combining centres of native globular 

 protein antigens. Other work has suggested that native globular proteins 

 will retain the power to combine with specific antisera after at least slight 

 enzymic hydrolysis (Landsteiner and Chase, 1933; Holiday, 1939; Klecz- 

 kowski, 1945; Lapresle, 1955a, b). 



We have therefore investigated the problem further and have found thats 

 if crystalline bovine serum albumin is hydrolysed by chymotrypsin in a 

 dialysis sack suspended in water brought to pH 8 with NH3 at 25°, then 

 material appears in the outer liquor (the diff"usate) which will specifically 

 inhibit the combination of bovine serum albumin with its antiserum. Full 

 details of this work have been given elsewhere (Porter, 1957). 



It was found that the active material (subsequently referred to as the 

 inhibitor) would not pass through a diff'erent sample of dialysis tubing and 

 hence after concentration in a rotary still it was redialysed in this second 

 sack with a smaller pore size. About half the weight was lost without loss 

 of activity. It was subsequently purified by ethanol precipitation and zone 

 electrophoresis and finally by precipitation with 5% trichloroacetic acid. 

 The precipitate redissolved in water and there was no loss of immunological 

 activity, but small adsorbed peptides were removed. 



The material now appeared to be pure. Electrophoresis and ultracentri- 

 fugation showed only one component and end group assay showed the 

 presence of only A^-terminal amino acid — phenylalanine — present as 1 mole 

 per 14,000. 



Estimation of molecular weight by sedimentation gave a value of about 

 24,000 — much higher than had been expected — and it was found that the 

 purified inhibitor would no longer pass through the large pore size dialysis 

 tubing as it had done in the first stage of preparation. It was eventually shown 

 that dimerization had occurred during purification and that this could be 

 reversed by cysteine at pH 4. Under these conditions the sedimentation co- 

 efficient corrected to water at 20° fell from 2-13 to 1-42 and the calculated 

 molecular weight from 24,000 to 12,000. The latter figure is close to the 



