Quantitative Spreading of Fibrinogen in Unimolecular Films

Abstract

Two general methods have been employed to spread proteins on aqueous surfaces in unimolecular films. Gorter and Grendel introduced the method of blowing the protein solution horizontally upon the surface of the water in a Langmuir trough. Hughes and Rideal placed on the water surface solid particles of the protein previously weighed on a microbalance. They could show that the films so obtained, while fluid at the outset, became elastic upon compression (sol-gel transition). Not all proteins, however, are amenable to spreading; examples are fibrinogen, and nerve nucleoprotein. These proteins spread very slowly and cannot be induced to form sol, i. e., fluid films. Instead, small amounts float about in elastic patches with equilibrium surface pressures of less than 0.1 dyne/cm., as compared with a pressure of 16 dynes/cm. for gliadin (Hughes and Rideal). The tendency to spread is so slight that because of its greater specific gravity the protein solution usually sinks into the buffer in the trough. It is highly desirable that methods be worked out for the quantitative spreading of such proteins. In attacking this problem we have worked with fibrinogen because of its ready availability and because methods for its purification are at hand. The fibrinogen was prepared according to the method of Hammarsten as modified by Florkin except that oxalated beef blood was used instead of citrated horse plasma. As recommended by Mellanby all salt solutions were oxalated; by this means the fibrinogen could be reprecipitated 3 times without denaturation. To apply the film we used a 0.2 ml. serological pipette graduated in thousandths. The pipette was sealed to one arm of a stopcock ground for vacuum; the other arm received a small hypodermic needle which was bent so that the protein solution was delivered horizontally with the pipette vertical. Films were spread on McIlvaine buffer, diluted tenfold. All pH determinations were made with the glass electrode.