Amino acid sequence of a fragment of rabbit muscle aldolase.

Cleavage of rabbit muscle aldolase by cyanogen bromide results in the formation of four fragments of different size [1]. Study of the primary structure of the enzyme has been based on the examination of these fragments [1, 2]. Because of the insolubility and associated problems due to the relatively large size of the fragments Lai [1], as well as Anderson et al. [3], used pyridine-acetic acid buffer as eluant for the Sephadex G75 column in the separation procedure. By combining cyanogen bromide cleavage and the reversible modification of amino groups we could successfully overcome these difficulties without impairing resolution [2]. Aldolase, carboxymethylated with 14C-bromoacetate in the presence of 8 M urea, was treated with cyanogen bromide as described by Lai [ 1 ]. The mixture of the fragments was acylated with maleic anhydride [4] or citraconyl anhydride [5] in the presence of 8 M urea, at pH 8.5. At the end of the reaction, the solution of the acylated fragments was directly applied to a Sephadex G-75 (fine) column. Elution was performed with 0.1 M ammonium bicarbonate (fig. I). The alignment of the four fragments separated by gel chromatography was first described by Lai [ 1 ] as CB I -CB3-CB4-CB2 and we confirmed his results by determining the Nand C-terminal sequences of the unblocked fragments. We further analysed the peptides of the tryptic and chymotryptic digest of acylated and deacylated CB 1, CB3 and CB4, as well as the chymotryptic, peptic and partial acid hydrolysate of the fragments obtained from the tryptic digest of the unblocked cyanogen bromide fragments. The N-terminal se-