•J. Soc. Cosmet. Chem., 28, 447-455 (August 1977) Studies on the molecular weight distribution of cosmetic protein hydrolysates ELAINE S. STERN and VERNON L. JOHNSEN Personal Care Division, Inolex Corporation, Chicago, IL. Received July 15, 1976. Presented Ninth IFSCC Congress, June 1976, Boston, MA. Synopsis MOLECULAR WEIGHT has been thought to be an important feature of COSMETIC GRADE PROTEINS and to be a critical factor affecting PROTEIN SUBSTANTIVITY to HAIR. The study reported in this paper was undertaken to show the relationship of molecular weight to protein substantivity. Using gel filtra- tion and ultracentrifugation data were obtained that indicate peptides in the range of molecular weight 1000 are more substantive than the very high molecular weight molecules. INTRODUCTION Cosmetic grade protein hydrolysates are complex mixtures of various molecular weight polypeptides. Only approximate number average molecular weights have been pre- viously reported. This study was undertaken to provide additional information about the distribution of molecular weights of collagen hydrolyzed by several methods, and to show if there is a relationship between molecular size and hair substantivity. Two techniques were used for investigating the molecular weight distribution of cos- metic grade protein hydrolysates, gel filtration and ultracentrifugation. Gel filtration is an established chromatographic method for the separation of molecules according'to their size. The use of Sephadex •, a bead-formed dextran gel, for gel filtration was introduced in 1959, and since has become a well-established method for fractionation and separation of molecules according to their size. Sephadex is cross- *Sephadex ©, Pharmacia Fine Chemical Inc., 800 Centennial Avenue, Piscataway, NJ. 447

448 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS linked dextran the degree of crosslinking determines the molecular weight fractiona- tion range. Fractionation Range (Molecular Weight) Sephadex Type Peptides Dextrans G-10 - 700 - 700 G-15 - 1,500 - 1,500 G-25 1,000- 5,000 100- 5,000 G-75 3,000- 70,000 1,000- 50,000 G-200 5,000-800,000 1,000-200,000 Molecules of molecular weight above the upper limit of the ranges shown in the above chart are totally excluded from the gel. Molecules of molecular weight below these ranges are eluted from the Sephadex column in the above chart at a volume about equal to the total bed volume. Molecules between the upper and lower limits are eluted from the Sephadex column in a specific relationship to the molecular weight. Over a considerable range, the elution volume is approximately a linear function of the logarithm of the molecular weight. The other method of determining molecular weight is by ultracentrifugation. The ultracentrifuge produces high centrifugal forces in order to measure the movement or redistribution of sedimenting particles. The distribution of the particles is observed by an interference pattern. From this interference pattern, a molecular weight or molecular weight range can be calculated. In work with the ultracentrifuge, the material under investigation is placed in the cen- terpiece of the cell assembly. This cell assembly is constructed in a manner that permits light rays to pass through its entire length. After the cell is assembled and placed in a rotor hole, the rotor is then installed in the rotor chamber the chamber is evacuated and the rotor accelerates. The sample material is subjected to high cen- trifugal forces that causes the molecular particles to sediment. As the particles are re- distributed, the light from the optical system light source can be transmitted through the rotating cell. By means of this light, the optical elements translate particle move- ment into an optical pattern, from which molecular weight can be calculated. METHODS AND MATERIAL MATERIALS Protein Hydrolysates 1. Collagen hydrolyzed with papain to a formol nitrogen of 10.0* 2. Collagen hydrolyzed with steam to a formol nitrogen of 6.0* 3. Collagen hydrolyzed with acid to a formol nitrogen of 10.0' *Inolex Corporation, Chicago, IL.