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A method is presented for calculating the number of guanidine hydrochloride and water molecules bound to proteins in 6 m guanidine hydrochloride solution. By correct combination of these quantities, the preferential interaction parameter of solvent components with proteins can be calculated, and, in turn, accurate values of the apparent partial specific volumes of proteins in 6 m guanidine hydrochloride can be predicted. This method requires only the knowledge of the amino acid composition of the protein. Application of this method to literature data shows that the predicted values of the partial specific volumes of proteins render possible an accurate estimation of their subunit molecular weights from sedimentation equilibrium experiments in 6 m guanidine hydrochloride.

Calf brain microtubule protein (tubulin) was characterized chemically. Amino acid analysis and hydrogen ion titration in 5 m guanidine hydrochloride yielded a chemical composition similar to that of tubulin from other sources. The NH2-terminal residue was identified as methionine. When the protein was denatured and reduced in 8 m urea, two distinct protein bands were observed in gel electrophoresis in the presence of urea, suggesting the existence of two nonidentical subunits. The molecular weight of the tubulin subunits was determined by a variety of techniques, including sedimentation equilibrium and light scattering in 6 m guanidine hydrochloride, sodium dodecyl sulfate gel electrophoresis, chromatrography on an agarose column in the presence of 6 m guanidine hydrochloride, and viscometry of S-carboxymethylated, reduced protein in 6 m guanidine hydrochloride. All methods yielded an average molecular weight value of (54,000 ± 1,000), whether the measurements were done with or without reducing agents, indicating that all disulfide bonds present are intrachain.

A method is presented for the determination of cholesterol and phospholipid, which requires 5 µl. of human serum or 1-2 ml. of cerebrospinal fluids. With this method 5-100 µg. of cholesterol and phospholipid can be separated by a modified silicic acid column after elution of the mixture with 1 ml. of chloroform and 3 ml. of methanol. Recovery for 24.6 µg. of cholesterol and 30.5 µg. of phospholipid was 98.4 and 96.7%, respectively. Standard deviations of ± 1.73 and ± 1.24 have been obtained for the reproducibility of cholesterol and phospholipid determinations after chromatography. The method has been applied for the estimation of the cholesterol/phospholipid ratio and of lipid phosphorus in total phosphorus of human cerebrospinal fluids.

A sucrose solution has been used as a stabilizing medium for the colchicine-binding activity of microtubule protein isolated from calf brain. The sucrose itself is shown not to interfere with the binding ability and the protein could be stored below 0 °C in the sucrose solution for at least 2 weeks without any significant loss of colchicine-binding activity.

A procedure has been developed to obtain a purified form of horse serum butyrylcholinesterase (BuChE) from a partially fractionated commercial preparation. A high recovery (greater than 80%) of the activity is achieved in each step of the purification procedure. In all steps involving column chromatography the cholinesterase activity is confined to a single, symmetrical activity peak. A single component is observed in the analytical ultracentrifuge over a 250-fold range of protein concentration (4–1000 μg/ml); the sedimentation coefficient, s 20,w 0 , is 11.5 ± 0.3S. There is no indication of molecular aggregation or dissociation. The purified material consists of a single migrating species upon polyacrylamide gel electrophoresis in gels with different pore sizes and at neutral and alkaline pH. Kinetic data indicate that the enzyme exhibits normal Michaelis-Menten behavior. K m for acetylcholine at pH 7.5 and 25 ° is 1.6 × 10 −3 m and the Hill coefficient is one. Mg 2+ and Ca 2+ exert a stimulatory effect on the enzyme activity. It is concluded that the purification procedures employed yield a homogeneous form of BuChE, which is not contaminated by other detectable proteins.