REVERSIBLE REDUCTIVE ALKYLATION OF AMINO-GROUPS IN PROTEINS

Amino groups of proteins can be alkylated by treatment with a carbonyl compound and a reducing agent [Means, G. E., & Feeney, R. E. (1968) Biochemistry 7, 2192], We are now reporting on a reversible method of reductive alkylation in which the amino groups are first alkylated by treatment with an α-hydroxy aldehyde or ketone in the presence of sodium borohydride. Since the chemical grouping RNHCH2C(OH)< (R=alkyl) is readily attacked by periodate to give the primary amine (RNH2), this modification is effectively reversed by treatment with periodate. Reversal of the modification is effected by treating the modified protein with relatively low levels of NaIO4 (10-20 mM) for ~30 min and is accompanied by a full or partial recovery of the activity lost as a result of modification. The best results were obtained with glycolaldehyde (HOH2CCHO) and with acetol (HOH2 CCOCH3). Glycolaldehyde reacts readily with amino groups to give modification at a high level but has the disadvantage that a second mole of the aldehyde can add relatively easily to the amino groups, giving a tertiary amine which is resistant to attack by periodate. With sodium borohydride as the reducing agent, ~20% of the amino groups modified by extensive treatment are converted to this irreversibly modified form. Higher levels of dialkylation are observed when sodium cyanoborohydride is used. Acetol, a ketone, is less reactive; with sodium borohydride as the reducing agent, only one molecule reacts with each amino group. Again, sodium cyanoborohydride can cause significant irreversible modification by favoring dialkylation of amino groups. The method was applied to four selected proteins: lysozyme, which is known to be rapidly inactivated by periodate; turkey ovomucoid, which is a trypsin inhibitor with an essential lysine residue and contains 25% carbohydrate; chicken ovomucoid, which is very similar in general structure to the turkey ovomucoid, but which has an essential arginine residue instead of a lysine; ribonuclease, in which the amino groups are essential for activity. © 1979, American Chemical Society. All rights reserved.