Abstract
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 mµ was studied as a function of temperature up to 80° or 85°. At these highest temperatures, the apparent order of stability was unreacted myoglobin g carboxamidomethyl derivative g carboxymethyl derivative, and aggregation with precipitation developed in a few minutes. The unreacted protein showed little or no tendency to redissolve on recooling to 23°. However, both derivatives were recovered to the extent of about 60% as apparently native protein. It was concluded that although electrostatic factors probably played a discernible role in determining the relative stabilities of the proteins at extremes of pH, such factors were not prominent in affecting the conformational transitions at high temperatures. Either modified derivative proved much better able to recover the native conformation on cooling than the unreacted protein. It is suggested that the advantage in having the external histidine residues modified may lie in the coding introduced to specify that such residues shall remain at all times in a hydrophilic environment, and not become trapped in erroneous, internal conformational variants.
Highlights
The carboxamidomethyl derivatives present in the sequence of Reaction 5 were analyzed after the acid hydrolysis as carboxymethyl derivatives
Narrowing of Limits of Stability by A lkylation-The results presented here confirm that the alkylation of ferrimyoglobin can be carried to the stage of modification of 6 or 7 histidine residues without the loss of native properties in neutral solution at room temperature (l-5)
The changes seen in alkaline solution were reversed by readjusting the pH to values below 10
Summary
Myoglobin was isolated by fractionation at low temperatures in ethanol-water mixtures containing metal salts [6]. After further fractionation with ammonium sulfate, the contamination. Stability of Alkylated Myoglobins Vol 242, No 14. Of the ferrimyoglobin with non-myoglobin proteins was less than 1% [6]. The individual myoglobin components were not separated from each other. Amino acid analysis followed previous methods [2, 7, 8]. Polyacrylamide gel electrophoretic analysis followed the method of Raymond [9]. Measurements of pH followed previous methods [6]. Spectrophotometric measurements were made with the Cary model 14 instrument
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