Effect of Alkylation of Sperm Whale Myoglobin on Response to Extremes of Temperature and pH

Abstract

Carboxymethyl and carboxamidomethyl derivatives of sperm whale ferrimyoglobin were prepared by reaction near pH 6.7 with bromoacetate and iodoacetamide, respectively.As had been established earlier, unless the reaction was carried to an extreme stage in the modification of histidine residues, the derivatives obtained were stable at neutral pH.The derivatives were generally less stable than the unreacted protein at high pH values, as judged by measurements of absorption and optical rotatory spectra.Of the two types of derivatives, the less highly negatively charged carboxamidomethyl myoglobin proved the more stable at pH 11.5 and above.In acid solution at pH 3.2 or pH 2.2, the derivatives, like the unreacted protein, showed a characteristically fully altered absorption spectrum.Optical rotatory measurements showed, however, that the apparent helix content decreased between pH 3.2 and pH 2.2.The decrease was least marked with the less strongly positively charged carboxymethyl myoglobin preparations.Both types of derivative behaved much like the unreacted protein in experiments in which the optical rotation at 233 rnp was studied as a function of temperature up to 80" or 85".At these highest temperatures, the apparent order of stability was unreacted myoglobin > carboxamidomethyl derivative > carboxymethyl derivative, and aggregation with precipitation developed in a few minutes.The unreacted protein showed little or no tendency to redissolve on retooling to 23".However, both derivatives were recovered to the extent of about 60% as apparently native protein.It was concluded that although electrostatic factors prob-