The thicknesses of hemoglobin and bovine serum albumin molecules as unimolecular layers adsorbed onto films of barium stearate.

The following work, which describes a method of measuring one dimension of some protein molecules, is based on the determination of the apparent thickness of a unimolecular layer of globular protein molecules adsorbed from solution onto a metallic slide covered with an optical gauge of barium stearate. Langmuirl' 2 and Rothen3 have published a few results obtained by such a technique, but have not exploited the method thoroughly. A complete set of experimental data has been obtained by Clowes4 on insulin and protamine. He studied the effects of pH and time of exposure on the thickness of layers of protamine and insulin adsorbed onto slides covered with barium stearate and conditioned with uranyl acetate. He found that the pH was responsible for large variations in the thickness of the adsorbed layers and that the thickness of insulin layers adsorbed onto a protamine base was dependent on the concentration of the insulin. Since Clowes found thicknesses as high as 100 A. for protamine and 400 A. for insulin, he was without doubt usually dealing with multi-layers. Experimental.-The apparent thickness of a protein layer is measured with an optical instrument called the ellipsometer by Rothen,5-7 who has given a complete description of its design and optics and has calculated its sensitivity as 0.3 A. This instrument measures the ellipticity of light reflected from a metallic slide when it is covered with a thin film of transparent material. The parameters of the ellipse of polarization are determined in part by the thickness and refractive index of the transparent layer of material on the slide. The ellipsometer, which is a type of polarim-eter, uses the half-shadow technique, by which a change in the ellipticity of the reflected light requires a change in angular setting of an analyzer used to balance the intensity of the half fields., As actually used, the instrument is calibrated with films of barium stearate, which for different known thicknesses, previously determined by x-ray diffraction measurements , require different angular settings of the analyzer for equal intensity of the half fields. In this way one measures the angular change in the analyzer setting produced by a film of unknown thickness and relates it by a calibration curve to a known thickness of barium stearate.