Amino acid analysis on polyvinylidene difluoride membranes.

A procedure for the amino acid analysis of proteins electrotransferred to polyvinylidene difluoride (PVDF) membranes is described. The proteins are first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a PVDF membrane. After staining with Coomassie brilliant blue, the visualized protein bands are excised from the membrane. Each band is placed in a vial and subjected to gas-phase hydrolysis in 6 N HCl in a vacuum desiccator at 110 degrees C. The amino acids are extracted from the membrane into 0.1 N HCl/30% CH3OH and analyzed by reverse-phase HPLC using postcolumn o-phthalaldehyde-derivatizing reagent. The method was shown to give reproducible and reasonably accurate compositions for several proteins, as well as to provide an estimate of protein content. As little as 10 pmol of a 67-kDa protein can be determined.

Solute and mobile phase contributions to retention in hydrophobic interaction chromatography of proteins.

Hydrophobic interaction chromatography utilizes high salt concentrations mobile phases to induce an interaction between a weakly hydrophobic matrix and exposed hydrophobic amino acids of a native protein. Proteins with a hydrophilic exterior have shorter retention times on a hydrophobic interaction column than do proteins with more hydrophobic exteriors. To examine the effect of amino acid substitutions on protein retention, lysozyme isolated from related bird species was chromatographed on a hydrophobic interaction column at increasing ammonium sulfate concentrations. Chromatographic retention deviated only when amino acid substitutions occurred on the surface of lysozyme opposite the catalytic cleft. This area may constitute a contact surface area and extends from Residue 41 to 102 and from 75 to the alpha-helical region starting with Residue 89. Retention was analyzed by plotting log k' versus the molal concentration of ammonium sulfate. The slope did not deviate significantly for each of the bird lysozymes, indicating a similar contact surface area. However, there was significant deviation in the intercept of each of the lysozyme lines, which probably reflects the strength of the hydrophobic interaction. The intercept increased as the lysozyme became more hydrophobic. Hydrophilic amino acid substitutions affected retention as much as hydrophobic ones. The ionization state of histidine residues within the contact area between lysozyme and the column surface also influenced retention. An uncharged histidine residue increased retention, while a decrease in retention was seen with a charged histidine residue. The amino acid substitutions did not appear to affect the size of the hydrophobic contact surface area, but rather the strength of the hydrophobic interaction. The effect of salt composition on protein retention indicated that factors other than surface tension could influence retention. These factors appear to include protein hydration and specific interactions between the protein and the salt ions. Of these, the latter may or may not result in an alteration in protein structure. The magnitude of the effect of salt composition was found to be dependent upon the protein.

Comparison of hydrophobic-interaction and reversed-phase chromatography of proteins.

The variable hydrophobic nature of proteins allows their separation through differential hydrophobic surface interactions. From these observations two modes of protein chromatography have been developed, hydrophobic-interaction chromatography (HIC) and reversed-phase chromatography (RPC). Selectivity of the HIC column can be easily manipulated by changing mobile phase variables. Protein retention was increased by decreasing the pH from neutrality or by using a salt with a greater "salting-out" ability. In addition, selectivity can be altered through chemical modification of the matrix surface. Protein retention and resolution decreased concomitantly with matrix ligand density. There were several major differences in HIC and RPC selectivity. Hydrophilic proteins such as cytochrome c and myoglobin were weakly retained on the HIC column but strongly retained on the RPC column. In contrast, a hydrophobic protein such as beta-glucosidase was strongly retained on the HIC column and only weakly retained on the RPC column. Other proteins were retained equally by RPC and HIC columns. Load capacity on the HIC column was determined by plotting resolution as a function of protein load. Resolution decreased significantly after 7.5 mg of total protein had been loaded onto the column per cm3 of column material. Samples of lactic dehydrogenase and alpha-chymotrypsin ranging in size from 10-200 micrograms were recovered from an HIC column with greater than 86% enzymatic activity in all cases. The recovery of enzymatic activity of alpha-chymotrypsin ranged from 55-91%, while none of the activity of beta-glucosidase was recovered from the RPC column.

High-performance hydrophobic interaction chromatography of proteins.

A new, weakly hydrophobic, high-performance liquid chromatography column has been developed for the separation of native proteins based on their relative hydrophobicities. Starting with a covalently bound, hydrophilic polyamine matrix, packing materials were synthesized through acylation with anhydrides and acid chlorides of increasing chain length to obtain increasingly hydrophobic surfaces. Proteins in aqueous buffers were induced to bind hydrophobically to the columns by the use of high salt concentrations in the mobile phase. Elution was achieved by decreasing the ionic strength of the solvent in a linear gradient. A mixture of cytochrome c, conalbumin, and beta-glucosidase was used as a standard to test the resolving power of newly synthesized columns. On a 4-cm butyrate column, baseline resolution was achieved in 20 min with a gradient of 3.0 mu sodium sulfate in 0.1 M potassium phosphate buffer, pH 7.0, to water. The static loading capacity for each column was determined using a hemoglobin binding assay. Capacities normally ranged between 150 and 180 mg of hemoglobin per gram of support. Since proteins are not denatured in hydrophobic interaction chromatography, enzymes eluted from the column retained enzymatic activity. Samples of alpha-amylase and beta-glucosidase ranging in size from 10 to 200 micrograms were recovered from the butyrate column with greater than 92% enzymatic activity in all cases. In a single trial, the enzyme citrate synthase was recovered from the benzoate column with 92% retention of enzymatic activity.

Calorimetric studies on monomeric and polymeric actin.

Differential scanning calorimetry of polymeric F-actin at pH 8.0 showed that the polymer had a concentration-independent thermal profile with a single transition temperature of 81 degrees C. In contrast, the thermal profile of G-actin was concentration-dependent, and although it resembled the F-actin profile at lower concentrations, it was found to have a more complex profile at higher protein concentrations.