Estimation of Molecular Weights of Protein-SDS Complexes

Barnett, W.A.; Spragg, S. P.

doi:10.1038/newbio234191a0

Cited by 8 publications

(3 citation statements)

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“…The trend in the estimates of the molecular parameters (Table 3) reflected the complex equilibria between myoglobin, free detergent and its micelles. The sedimentation-equilibrium experiments (Barnett & Spragg, 1971) support the present estimates of molecular weights for myoglobin-detergent complex, which in turn show that the partial specific volume of myoglobin in sodium dodecyl sulphate is between 0.80 and 0.82ml/g. The relationship between the sedimentation coefficient and 1/(time) follows the pattern predicted by Baldwin (1954) for heterogeneous mixtures and differs both in sign and magnitude of the slope from that observed for pure myoglobin in sucrose (see Fig.…”

Section: Discussionsupporting

confidence: 80%

“…128 was mixed with 2 % (w/v) sodium dodecyl sulphate by adding weighed amounts of sodium dodecyl sulphate to the myoglobin solution. Centrifuge equilibrium experiments with this mixture have shown that the estimate of the molecular weight of myoglobin under these conditions is low (Barnett & Spragg, 1971). It is thought that the low value reflects the formation of polymicelles with the protein, which were absent from the sodium dodecyl sulphate solution alone.…”

Section: Xs (S)mentioning

confidence: 90%

“…and it seems from the work of Reynolds & Tanford (1970), and confirmed for myoglobin by Barnett & Spragg (1971), that a more realistic value for the partial specific volume would be 0.82ml/g. If this partial specific volume is used to correct the extrapolated value from Fig.…”

Section: Xs (S)mentioning

confidence: 91%

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The statistical estimation of molecular weights from normal and difference ultracentrifuge boundaries

McCallum

Spragg

1972

Biochemical Journal

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1. To determine molecular weights from boundary data taken from a sedimentation velocity experiment in an ultracentrifuge, the parameter s/D must be estimated. This can be obtained by using non-linear statistical methods to fit a mathematical model [the Fujita & MacCosham (1959) equation] to the results. 2. The statistical method chosen was the simplex method of Nelder & Mead (1965), which was found to be ideal for this problem. Internal errors were calculated at the end of the search for the minimum in the residuals, but in general these errors were found to not represent the overall true error of the experiment. 3. Calculations of molecular weights of myoglobin showed that instabilities at low concentrations of protein (less than 0.8mg/ml) disturbed the calculation of s/D. If 1% (w/v) sucrose was included in the solvent, these instabilities were decreased, and extrapolating to infinite time the linear function of s versus 1/(time) gave an acceptable value for s with an error of +/-4.8%. The estimates of the molecular weights were less well-defined and the mean value was low by 8%, with an estimated error of the mean of +/-3%. The conclusion was that vibration was responsible for the instabilities without sucrose. 4. The Fujita-MacCosham equation can be extended to make it possible to estimate ratios of sedimentation and molecular weights for difference boundaries. Tests using two solutions of orosomucoid in which a 2% decrease in velocity of one boundary was achieved by adding a calculated quantity of sucrose showed that the analysis gave realistic values for the two ratios, and the error for the ratio of sedimentation coefficients was +/-10%. The error was larger for the estimated ratio of the molecular weights, but the analysis gave the expected value for the ratio.

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Section: Discussionsupporting

confidence: 80%

Section: Xs (S)mentioning

confidence: 90%