Characterization of the reaction of methyl acetimidate with sperm whale myoglobin

Abstract

The effects of pH, acetimidate concentration, temperature, and reaction time of methyl acetimidate with sperm whale myoglobulin have been assessed. Reaction at pH 9.8 and 15 degrees C for 30 min with a sixfold excess of methyl acetimidate relative to each amino group yielded six acetimidomyoglobin derivatives which were separated and purified. Reaction with tetrahydrophthalic anhydride revealed the number of amino groups that remained unreacted in each separated component and made possible further subractionation. Modification at the NH2 terminus was quantitated by automated stepwise Edman degradation. The acetimidyl and tetrahydrophthalyl groups, were readily removable. The potentiometric titration of three of the completely deprotected components showed identity with the parent untreated sperm whale myoglobin. The first of two major products was acetimidated at all 19 epsilon-amino groups but not at the NH2 terminus. The second major product bore a blocked NH2 terminus but retained one unmodified epsilon-amino group, identified after modification by trinitrobenzenesulfonate as lysine residue 77. Of the minor components, one was identified as completely acetimidated at all 20 amino groups. The other three minor components appeared to contain irreversible by-products.

Semisynthesis of sperm whale myoglobin by fragment condensation.

“…Sperm whale myoglobin was isolated, and the major component band IV (7) was purified (8). Preparation of acetimidomyoglobin and its reaction with tetrahydrophthalic anhydride to isolate the Na,NE -acetimidyl derivative follow the procedure of DiMarchi et al (1). Upon removal of the heme (9), the apoprotein was dissolved (3%) in 50% (vol/vol) acetic acid/6 M urea, and a 180-fold molar excess ofphenol was added.…”

“…The mole fraction ofnascent protein fragments within a sample ofcleavage or coupling products was determined by subjecting the unfractionatedproducts to several degradative cycles on the Beckman 890C sequencer directed by the Beckman fast protein Quadrol program (072172C); the NH2-terminal residues were identified as described (1). The recovered amino acids were correlated with the fragment of origin.…”

“…Studies using selective, consecutive removal and substitution ofthe NH2-terminal residues ofMb by specific degradation and resynthesis (1,2) have indicated the subtle contribution of the NH2-terminal charge and the uncharged side chains ofresidues 1 and 2 to the conformation and stability of the native molecule (3). To examine further the effects of synthetic sequence alteration with respect to stability, conformation, and electrostatic and hydrophobic interactions, a method for creating internal sequence variations must be developed.…”

“…The current strategy exploits the oxidative lability of the tryptophans at positions 7 and 14 ofthe Mb sequence by the use of3-bromo-2-(nitrophenylsulfenyl)skatole (BNPS-skatole; skatole = 3-methylindole) to effect cleavage, yielding the fragment corresponding to residues 15-153 (designated fragment in which the E-NH2 groups have been protected previously by reaction with methyl acetimidate (1). Sequential coupling of the N-hydroxysuccinimide (HOSu) esters of o-nitrophenylsulfenyl-L-tryptophan (NPS-Trp; residue 14) and the selectively protected peptides corresponding to residues 1-5 and 6-13 (designated peptides 1-5 and 6-13) regenerated the native sequence.…”

Site-specific semisynthetic variant of human hemoglobin.

“…Preparation and purification of the p-hydroxymercuribenzoate-labeled Nasulfophenylthiocarbamoyl-a chains (Na-SPTC-PMB-a chains) was described above. Protection of e-amino and sulfhydryl groups with methyl acetimidate hydrochloride (19) and p-hydroxymercuribenzoate, respectively, was necessary to direct coupling of the N-hydroxysuccinimide ester of CF3CO-glycine (CF3CO-Gly-ONSu) to the NH2-terminal position. Purification of the totally protected (N'11-acetimido-Na-SPTC-PMB)-a chains was accomplished by anion-exchange chromatography following reaction of the acetimido-Na-SPTC-PMB-a chains with 3,4,5,6-tetrahydrophthalic anhydride (19) to introduce a negative charge on free NH2 groups.…”