This treatment had no effect on the binding activity of the protein.Combination of the altered cyanocobalamin molecule with SF. The ultrafiltration method of measuring binding activity showed that the monobasic acid E, the monobasic acid without the cyanocobalamin nucleotide, the tribasic acid, thiocyanatocobalamin and formaldehydetreated all combined equally with SF. In factor A, the dimethylbenziminazole moiety of cyanocobalamin is replaced by methyladenine and in pseudovitamin B it is replaced by adenine. Removal of the nucleotide from cyanocobalamin, factor A or pseudovitamin B gives factor B. The nucleotide group, therefore, is not concerned in the combination of the vitamin with the protein.The experiments reported in this paper have shown further that <a href="http://www.targetmol.com/compound/Rasagiline-Mesylate">Targetmol's
Rasagiline Mesylate</a> hydrolysis of the three primary amide groups in the cyanocobalamin molecule does not affect the ability of the vitamin to combine with the protein, but more drastic hydrolysis to the hexa and hepta acids gave a substance which no longer combined with the protein.The experiments with cobalamin showed that the protein did not displace the cyano group when it combined with cyanocobalamin, and since thiocyanatocobalamin also combines with the protein, the linkage does not take place through the cyano group.This fact suggests that either the protein is linked to the vitamin in the place where the second cyano group normally adds on, or that steric hindrance by the protein prevents the cyanide ion from adding on to the cyanocobalamin in the complex.A more precise determination of the point of attachment of the protein to the vitamin molecule now awaits further information about the core of the cyanoco balamin molecule.The group on the protein molecule, concerned with its combination with cyanocobalamin, is not a sulphydryl group, since iodosobenzoic acid, iodoacetic acid, iodine acting as an oxidizing agent and pchloromercuribenzoate had no effect on the binding activity of SF.Formaldehyde at pH reacts with amino groups on proteins, and at pH it attacks both amino and amido groups. At neither pH did it affect the combination of SF with cyanocobalamin.The acetylation of SF with this reagent at both pH and had no effect on its combination with cyanocobalamin.If an amino group is concerned, it must be one that is not readily acetylated by this reagent or attacked by formaldehyde.Fluorodinitrobenzene and iodine, at the higher concentrations used, were the only reagents that prevented the combination of SF with the vitamin.Fluorodinitrobenzene, under the conditions used in these experiments, attacks amino and phenolic groups and to a lesser extent imidazole and sulphydryl groups. Iodine acts as an oxidizing agent at an acid pH, in the presence of a high concentration of KI.With a low concentration and a higher pH, the oxidizing action of iodine is largely suppressed and iodination occurs.Under these conditions, iodination of the protein prevented its combination with the vitamin, and therefore it is possible that a phenolic group on the protein may be concerned in its linkage with cyanocobalamin.Its amino acid composition is typical of proteins, except for a high tyrosine content.If a constant supply of material was applied at one place on the top surface it was carried down by the buffer solution to the outlet immediately below.