Popp/Baiier/t hasetti/CONnRaD
210
Blood donors were composed of 3 laboratory personnel, [2 Marshall
Island residents, and 13 Marshall Island residents exposed to y-radiation
fallout in 1954; the latter were positive controls. 10 ml of blood from each
individual was collected in a heparinized syringe. Erythrocytes were separated
from plasina by centrifugation for 10 min at 600 g and washed 3 times in
10 vol ofsaline. Samples from the Marshallese were resuspended in an equal
volume of phosphate-buffered saline and shipped on ice by air and washed
again uponarrival. The packed red cells were lysed by adding 4 vol of cold
distitled water. Red cell stroma was removed by centrifugation for 15 min
at 20,000 g. Hemoglobin in the supernatant was converted to the carbon
monoxide form by bubbling with CO in a hood; one drop of octanol was
added to prevent foaming. The hemoglobin solutions were kept coid during
all subsequent processing (cold room at 4°C),
S012b;5._.
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Materials and Methods
sm taineg
hg
Because human hemogtobin A, in comparison with most other proteins,
has an unusual chemical property, i.e. it contains no coded isoleucine {3},
we believed that chemical methods alone could be used to determine the
substitution frequency of isoleucine for other amino acids in highly purified
hemoglobin fromindividuals of various ages. An increase in the isoleucine
substitution frequency with age could be evidence in support of the error
theory of ageing. However, the small quantity of isoleucine found in hemoglobin A can be incorporated by more than one mechanism. These are:
(I) infrequent errors in the transcription of DNA to mRNA may change
nonisoleucine codons into isoleucine codons; (2) errors in transcription may
produce allered forms of tRNA; these may have reduced fidelity as regards
both the kinds of amino acids they accept and the species of mRNA codons
they recognize: (3) errors in translation of the correct mRNA can occur
through mispairing of tRNA anticodons and their proper mRNA codons;
(4) amino acy! synthetase errors can cause the wrong amino acid-tRNA
complexes (o be formed, and (5) mistakes in the replication of DNA involving the exchange of appropriate base pairs [2] lead to the formation of mutant
cells whose hemogtobin mRNAscontain isoleucine codons. Chemical determinations, as a composite measure of these errors, of the frequency at which
isoleucine substitutes for ather amino acids in hemoglobin of normal adults,
ages 20-5], are reported in this paper.