Studies of an associating protein system: Sub‐unit molecular weight of the low sulphur fraction of soluble wool keratin

Abstract

Solutions of the low-sulphur fraction of soluble S-carboxymethyl wool keratin in 60 yo formamide were previously shown to dissociate on dilution. In order to obtain the molecular weight of the smallest sub-unit present, data are reported using the techniques of osmometry and equilibrium centrifugation a t high speed. Values for Mn and M, a t infinite dilution obtained by both techniques lie in the range 23,000-25,000. The system as a whole probably involves an equilibrium between monomer, dimer, and trimer and the relation of this to the structure of the crystalline part of the wool fibre is discussed. ZUSAMMENFASSUNG:I n einer friiheren Arbeit uber losliches S-Carboxymethyl-Wollkeratin wurde gezeigt, da13 in der Losung der schwefelarmen Fraktion in 60-proz. Formamid bei Verdunnung Dissoziation stattfindet. I n der vorliegenden Arbeit wurden die Molekulargewichte der kleinsten Untereinheit auf osmotischem Wege und aus dem Sedimentationsgleichgewicht bestimmt. Dabei lagen die Werte fiir das Zahlenmittel Mn und das Gewichtsmittel M, bei unendlicher Verdiinnung im Bereich 23 000-25 000. Das geloste System enthat wahrscheinlich ein Gemisch aus Mono-, Di-und Trimeren, die miteinander im Gleichgewicht stehen; dies wurde im Zusammenhang mit der Struktur des kristallinen Teils der Wollfaser untersucht.

Studies on Reduced Wool V. A Comparison of the Two Major Components

“…This can only be assessed if the molecular weight estimated by gel-filtration behaviour is confirmed by classical physicochemical techniques which are not subject to these uncertainties. Themolecularweight of component 8 determined from ultracentrifuge data at normal concentrations accords well with the estimates made from its behaviour on Sephadex columns at similar concentrations and this estimate by gel-filtration did not change at much lower concentrations, even below the concentrations used in the experiments of DeDeurwaerder and Harrap (1965). However, these authors used a different starting material (SCMKA) and Harrap (personal communication) has obtained, by osmotic pressure measurements, values for the molecular weight of our isolated components separated on Sephadex columns consistent with those reported in this paper.…”

“…used to see if we could gain any evidence from elution volume values, as distinct from the shape of curve discussed previously, to support the results of DeDeurwaerder and Harrap ( , 1965. Their results have been interpreted as showing the existence of a reversible dissociation of SCMKA at low protein concentrations in 14M formamide to give a subunit of molecular weight approximately 25,000.…”

“…There is some evidence which suggests the existence of subunits in components 7 and 8. , using a surface balance, has found minimum values of about 8000 for the molecular weights of fractions of low-sulphur proteins from wool. Furthermore DeDeurwaerder and Harrap ( , 1965 have presented evidence from sedimentation and osmotic pressure data that there is a dissociation of SCMKA to subunits of approximately 25,000. It should be pointed out that SCMKA is less pure than components 7 and 8, as it contains, in addition, aggregated material and some high-glycine-high-aromatic amino acid protein fraction with smaller amounts of high-sulphur proteins and unpublished data].…”

“…In order to obtain equal-sized effluent fractions, Beckman AccuFlo pumps were used to control the flow of buffer to the top of the column to a rate of 16 ml/hr. For studies of molecular weights by gel-filtration it was necessary to incorporate salt in the eluting buffer to minimize any non-ideality effects; the buffers used were 8M urea-O· 05M Tris-O ·IM KCI-O· OOIM Versene at pH 7· 4, 60% formamide-0'05M phosphate at pH 7 [similar to that used by Harrap (1964, 1965)], and 5M guanidine hydrochloride-0·01M Versene at pH values ranging from STUDIES ON REDUCED WOOL. V 1209 and were carboxymethylated in the same way at pH 8·5.…”

“…For studies of molecular weights by gel-filtration it was necessary to incorporate salt in the eluting buffer to minimize any non-ideality effects; the buffers used were 8M urea-O· 05M Tris-O ·IM KCI-O· OOIM Versene at pH 7 . 4, 60% formamide-0'05M phosphate at pH 7 [similar to that used by Harrap (1964, 1965)], and 5M guanidine hydrochloride-O'OlM Versene at pH values ranging from 7 to 8. In general, the methods used to measure elution volumes were similar to those of and .…”