[ 19521 Goring and Johnson. 9. An Ultrafiltration Method for the Optical Clurijication of Protein Solutions in Light -scattering Investigations. By D. A. I. GORINGand P. JOHNSON. In the application of the light-scattering method to molecular-weight and -size determinations of dissolved macromolecules removal of aggregated material and all impurity is essential. The more usual methods of filtration are inadequate and a simple ultrafiltration technique based on Elford’s Gradacol membranes has been evolved. By its use molecular-weight values in fair agreement with accepted values have been obtained as well as low and reproducible dissymmetry values. SINCEthe light-scattering method yields a weight-average molecular weight (M) for dissolved macromolecules erroneously large values are caused by the presence of relatively small amounts of heavy aggregates or impurity.In a current light-scattering investigation considerable difficulty was found in removing such aggregates from solutions of certain globulin-type seed proteins. For aqueous systems the ordinary filtration methods employing fine sintered discs Seitz or cellulose-pulp pads were ineffective ; high-speed centrifugation (12 000 r.p.m.) only partially cleared the solutions for it was found impossible not to stir up sediment when removing tubes from the rotor. An ultra- filtration technique based on the Gradacol membranes developed by Elford (Trans. Faraday SOL,1937,33 1094)was finally adopted with which clear solutions were obtained giving molecular weights near the accepted values.The light-scattering apparatus used is described elsewhere (Goring and Johnson publication pending). EXPERIMENTAL Membranes were made in the same way as the thimble-type osmometer membranes described by Adair (Proc. Roy. SOG.,1925 A 108 627) and Alexander and Johnson (“ Colloid Science,” Oxford Univ. Press 1949). A suitable nitrocellulose solution was poured evenly over a test tube-shaped mould perforated by a small hole at the rounded end ; the mould was rotated at about 15 r.p.m. Each coat was allowed to dry for a given time before the next was applied. No heat was used for drying and the temperature and humidity were not controlled. With Elford’s Gradacol membranes intended for gradation of particle size reproducible and uniform pore diameters are ensured by strict control of temperature and humidity in the drying process.Since the purpose here was merely removal of aggregates such care in preparation was considered unnecessary. After soaking in water overnight the thimble was removed by forcing water under pressure between the film and the glass through the perforation. High quality rubber pressure tubing was used for mounting and the final size of the membrane was ca. 40 mm. long by 10 mm. diameter giving a filter area of 12 sq. cm. Membranes were stored in 40% saturated aqueous ammonium sulphate and were cleaned for re-use with N/lO-hydro- chloric acid or 0.1yo sodium hydroxide solution. More concentrated solutions of alkali caused the nitrocellulose to become brittle and crack.Absolute ethanol sodium-dried ether and AnalaR acetone amyl alcohol and glacial acetic acid were used as solvents ; the nitrocellulose was a sample (grade HL 120/170) recommended by Alexander and Johnson for membrane formation. The most useful membranes for filtration of seed-globulin solutions (M ,” 300,000) were made by applying three coats of a solution of the following composition nitrocellulose (2 g.) ether (18 g.) alcohol (7 g.) acetone (9 g.) amyl alcohol (4 g.) and acetic acid (0.13 g.). As reported by Elford the pore size was found to be sensitive to the concentration of acetic acid and membranes with greater or smaller permeabilities were made by using respectively less or more acetic acid in the nitrocellulose solution. Times of drying before the second and the third coat were applied were 2 and 3 minutes respectively and for the final drying moulds were suspended in a vertical position for 1 hour before being soaked.The permeability of these membranes to water was 0.7 c.c./minute under a pressure of 5 cm. of mercury and was fairly reproducible ( -J= 10%). With protein solutions flow rates were slower because of the increased viscosity of the fluid and probably the blockage of the pores by large aggregates. By means of a mercury manometer protein solutions were forced through the membranes D Optical Clay@ cation of Protein Solutions etc. under pressures of 5-15 cm. Hg. If the solution was so cloudy that filtration proved very slow a preliminary filtration through a sintered glass disc was found preferable to the use of greater pressures which tended to pollute the solution.The proportion of material held up by the filter varied with the protein preparation the state of the solution and the ionic strength I. With serum albumin (I= 0.1) and edestin solutions (from hemp seed) (I = l) respectively the hold-up of material was 15% and 25% while for strongly aggregated solutions of arachin (from ground-nuts) (I = 0.1) less than half of the protein was found in the filtrate. Easy filtration with small hold-up was favoured by solutions of high ionic strength. For example with a solution of legumin (from peas) in phosphate buffer of pH 7-7 and I = 0.1 the hold-up was 75% falling to 20% when the ionic strength was increased to 0.5.This effect may have been due to either the increasing tendency to aggregation at low values of I (due to the lowered solubility of the globulin) or an increase in particle-membrane interaction at low ionic strengths. An example of the effectiveness of the filters is given in the figure which shows the normalised intensity distributions of scattered light for buffers cleaned respectively by filtration through Plot of Yn against 0 for buffer solutionsfiltered through a No. 4 " Pyrex " sintered-glass disc (I)and an ultrafiltration membrane (11). 0 1 u-" ao e-w I I I I a No. 4. Pyrex sintered-glass disc and by the present ultrafiltration technique. The normalised intensity 9nis given by Yn= 9sin e/(l + cos2 e) where 9is the observed intensity and 8 is the angle the scattered light makes with the transmitted beam.For the ultra-filtered buffer ynis constant and the dissymmetry 9600/~120. is virtually unity indicating that heavy material had been completely removed by the filter. The irregularities in the graph are due to small imperfections in the light-scattering apparatus which become apparent at such low values of 9n;here ynfor the ultra-filtered buffer is less than 1% of the scatter shown by a 0.3y0solution of arachin. With the buffer filtered by the sintered- glass disc Ynincreased markedly for smaller values of 8,and 960./9l20. was 2.7 showing the presence of large scattering particles in the buffer. As a further test solutions of serum albumin and arachin known to contain a considerable amount of aggregated material were filtered first through a No.4. " Pyrex " sintered-glass disc and then through a membrane. Light-scattering determinations of molecular weight were made after each filtration. From the results of these measurements shown in the Table it is Decrtase irt Molecular Weight and Dissymmetry after Ultrafiltration. -M 46 /412, A r 3c > Accepted found before found after before after Protein value ultrafiltration ultrafiltration ultrafiltration ultrafiltration Arachin ..................... 330,000 3,260,000 542,000 1.95 1.26 Serum albumin ............ 70,000 140,000 85,000 1-69 1.04 clear that much of the aggregated material passed through the sintered-glass disc to give high values of M and 960/Y,20 which were considerably reduced after ultrafiltration.The M values were still somewhat higher than the accepted molecular weights because strongly aggregated [I9521 The Reactiovt of Some SNgar Acetates with Ammonia. solutions were especially chosen to test the membranes. Freshly prepared solutions gave accurate values for the molecular weight of serum albumin; for the seed globulins arachin legumin and edestin molecular weights only 10% higher than the accepted values were noted. This discrepancy is not necessarily attributed entirely to the light-scattering determination. We are grateful to Mr. B. P. Brand for the preparation of the legumin samples used. DEPARTMENTCOLLOID SCIENCE, OF THEUNIVERSITY, CAMBRIDGE. [Received July 4th 1951.]