Amino Acids, Peptides and Proteins - Devényi T., Gergely J. 1976

Some methodological aspects of the analytical study of proteins
Study of native proteins
Membrane filtration

Membrane filtration, also known as ultrafiltration or molecular filtration, is a process for separating substances using membranes with a specific pore size. In recent years, membrane filtration has become widely used due to The production of a large variety of membrane types and specialized equipment. Membrane filtration is employed as a rapid and gentle method for removing a solvent from a macromolecular solution or for replacing one solvent with another. Most commonly, such tasks are encountered during the Desalting or concentration of macromolecular solutions. Another important application of membrane filtration is the Separation of two or more components that differ in their molecular size. Finally, molecular filtration allows for The Study of the binding of low-molecular-weight compounds by macromolecules.

Blatt's review lists the following primary requirements for membranes: they must have specific pore sizes, allow the solution to pass through at a sufficiently high rate, and exhibit minimal adsorptive capacity. Currently, several manufacturers produce filtration membranes that meet these requirements to varying degrees (Table 4). Anisotropic membranes, consisting of a dense, very thin selectively permeable Skin-membrane attached to a porous support, have become the most widely used. Table 4 indicates the Molecular Weight of substances retained by the membrane, but in practice, membranes do not retain 100% of the corresponding macromolecules, but slightly less. Consequently, for more complete retention, one should select a membrane with smaller pore sizes than those listed in the table. For example, for albumin (mol. wt. 67,000), it is better to use PM-30 rather than XM-50. It should also be noted that The ability to pass through a membrane depends not only on molecular weight but also on molecular shape and flexibility. In addition to those listed in Table 4, isotropic ultrafilters manufactured by Sartorius (FRG) should be mentioned; these are made of regenerated Cellulose (SM 115 series, pore size of different filters in the series is 150–5 nm), cellulose acetate (SM 117 series, pore size 35–5 nm), and cellulose nitrate (SM 121 series, pore size 15–5 nm).

¹ This section was written by R. S. Nezlin and included in the chapter by agreement with the authors. — Ed. note.

Class="center">Table 4 Membranes for ultrafiltration (Blatt, 1971)

One of the major problems in membrane filtration is The Development of a concentration gradient: at the very surface of the membrane, the concentration of macromolecules becomes so high that it can impede ultrafiltration. This is particularly noticeable when working with high-molecular-weight Proteins. To achieve good results in membrane filtration, the choice of equipment plays a crucial role.

Commercially available pressure filtration Cells with continuous stirring of the liquid near the membrane (stirred cells) are very simple to operate. Such convenient and reliable cells, manufactured by Amicon (USA) in several sizes (10, 60, 200, 400, and 2000 ml), have become widely used. They are made of Materials with low adsorptive capacity, are easy to assemble, and are highly durable when used carefully. Amicon membranes, which possess high selectivity, can be reused multiple times; if the liquid flow through them gradually slows down, Treatment with dilute protease solutions (e.g., Trypsin) restores their filtration capacity. A gas pressure (e.g., nitrogen) of 2–3 atm usually ensures a sufficiently rapid liquid flow. The cells can be placed in an ice bath; however, filtration is twice as slow in the cold.

The disadvantage of stirred cells is that they are rather difficult to use when dealing with concentrated protein solutions. Other, more complex systems prove suitable in these cases, and they offer higher filtration rates. These include devices with a thin-channel system above the membrane and devices with a hollow-fiber system (microtubular membranes) with lumen diameters of 0.2–0.5 mm. The high flow rate of the liquid in the channels prevents The formation of a concentration gradient at the walls. Increasing the filtration area sharply increases the flow rate, but at the same time, non-specific protein sorption increases, leading to significant losses when working with dilute solutions. Commercially available microtubular membranes from Amicon (USA) retain proteins with molecular weights of 10,000 (PM) and 50,000 (XM), and membranes from Bio-Rad (USA) retain proteins with molecular weights of 100,000 (Bio-Fiber 80), 30,000 (Bio-Fiber 50), and 5,000 (Bio-Fiber 20).

Recently, very simple systems for the single-use concentration of small-volume samples, known as Minicon concentrators (Amicon, USA), have also been introduced. These are flat, cone-shaped vessels with a membrane on one side, backed on the outside by a liquid-absorbing layer. The lower, pointed part of the vessel lacks a membrane, and the concentrated liquid accumulates there. This type of vessel requires no additional equipment (stirrer, gas cylinder) and is therefore particularly convenient in clinical laboratories for the rapid concentration of urine, CEREBROSPINAL FLUID, or other protein solutions prior to their subsequent analysis by Electrophoresis or other Methods. The following types of Minicon concentrators are commercially available, as listed in Table 5.

Table 5

References

1. Aronsson T., Grönwall A., Scand. J. Clin. Lab. Invest., 9, 338 (1957).

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Recommended reading

Blatt W. F., Methods in Enzymol., vol. XXII, Academic Press, New York, London, p. 39 (1971).

Clausen J., Immunochemical Techniques for the Identification and Estimation of Macromolecules, North Holland Publishing Company, Amsterdam, London, 1969.

Fischer L., Introduction to Gel Chromatography, North Holland Publishing Company, Amsterdam, London, 1969.

Freedman S. O., Clinical Immunology, Harper-Row, New York, 1971.

Gordon A. H., Electrophoresis of Proteins in Polyacrylamide and Starch Gels, North Holland Publishing Company, Amsterdam, London, 1969.

Kabat E. A., Einführung in die Immunochemie, Springer Verlag, 1971.

Leach S. J., ed., Physical Principles and Techniques of Protein Chemistry, Academic Press, New York, London, 1969.

Maurer H. R., Disc electrophoresis and Related Techniques of Polyacrylamide gel electrophoresis, Walter de Gruyter Berlin, New York, 1971.

Mollison P. L., Blood Transfusion in Clinical Medicine, Blackwell Scientific Publications, Oxford, London, Edinburgh, Melbourne, 1972.

Schultze H. E., Heremans J. E., Molecular Biology of Human Proteins, Vol.

I, Elsevier Publishing Company, Amsterdam, London, New York, 1966. Weir D. M., Handbook of Experimental Immunology, Blackwell Scientific Publications, Oxford, London, Edinburgh, Melbourne, 1973.

Williams C. A., Chase M. W., Methods in Immunology and Immunochemistry, vols. II—III, Academic Press, New York, London, 1968, 1971.