Characterization of the stable, acid-induced, molten globule-like state of staphylococcal nuclease.

Titration of a salt-free solution of native staphylococcal nuclease by HCl leads to an unfolding transition in the vicinity of pH 4, as determined by near- and far-UV circular dichroism. At pH 2-3, the protein is substantially unfolded. The addition of further HCl results in a second transition, this one to a more structured species (the A state) with the properties of an expanded molten globule, namely substantial secondary structure, little or no tertiary structure, relatively compact size as determined by hydrodynamic radius, and the ability to bind the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid. The addition of anions, in the form of neutral salts, to the acid-unfolded state at pH 2 also causes a transition leading to the A state. Fourier transform infrared analysis of the amide I band was used to compare the amount and type of secondary structure in the native and A states. A significant decrease in alpha-helix structure, with a corresponding increase in beta or extended structure, was observed in the A state, compared to the native state. A model to account for such compact denatured states is proposed.

Characterization of protein behavior in high-performance capillary electrophoresis using a novel capillary system.

Original calculations of over a million theoretical plate efficiency for macromolecular solutes in the open tubular high-performance capillary electrophoresis experiment considered axial diffusion to be the efficiency limiting factor. In practice, interactions of biopolymers, such as proteins, with the capillary wall has had a significant impact on readily achieving high efficiencies for a wide variety of proteins. This paper reports a capillary system in which protein-surface interactions have been minimized, resulting in high efficiencies (greater than or equal to 300,000 theoretical plates). This system allows the analysis of a set of protein standards over a wide pI range at neutral pH and moderate ionic strength. The characterization of the behavior of those protein standards in this capillary system is described.

Attenuated total reflectance Fourier transform infrared analysis of an acyl-enzyme intermediate of alpha-chymotrypsin.

Attenuated total reflectance Fourier transform infrared (FTIR) spectroscopy was used to obtain signal enhancement of the spectrum of the trans-cinnamoyl-alpha-chymotrypsin acyl-enzyme intermediate. Dilute solutions (as low as 2.5 mg/ml) of enzyme or stabilized acyl-enzyme intermediate were used to form thin films on a germanium crystal surface. The secondary structure of the enzyme thin film was shown to be consistent with the native secondary structure using deconvoluted FTIR data. A novel subtraction technique was used to eliminate interfering spectra of water vapor and protein in critical regions of analysis for esters. This permitted the difference spectra of the one new ester carbonyl bond to be discerned from the 300 or so amide bonds in the protein. The results suggest that the acyl-enzyme exists in two different conformations. This study demonstrates that ir structural information of enzyme-substrate or enzyme-inhibitor complexes can be obtained with dilute protein solutions.

Analysis of recombinant DNA-derived glycoproteins via high-performance capillary electrophoresis coupled with off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

This paper describes the analysis of glycoform populations of the glycoproteins ovalbumin and Desmodus salivary plasminogen activator (DSPA alpha 1) by a combination of capillary electrophoresis (CE) and off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Ovalbumin has a single N-linked glycosylation site and DSPA alpha 1 has six sites for potential glycosylation, 2 N-linked and four O-linked. The conditions used for the electrophoretic separation of ovalbumin include a borate buffer system, together with a diamine additive such as 1,4-diaminobutane (DAB). An electropherogram of DSPA glycoforms could be obtained at pH 3.0 (phosphate buffer) using a bovine serum albumin (BSA) coated capillary. Fraction collection was performed by controlled application of pressure [5000 Pa (50 mbar)] for zone elution and MALDI-TOF-MS was performed on samples prepared by a 1:1 dilution with the UV absorbing matrix sinapinic acid. Both electrophoretic separations were successfully characterized by good quality mass spectra and distinct mass trends were observed for the collected fractions. It is likely that each of the collected fractions are still mixtures of glycoforms and explanation of relative mobilities or masses of different fractions is not possible at this stage. The ability to perform rapid off-line MALDI-TOF-MS of fractions from complex electropherograms will be a powerful tool to demonstrate product consistency in the manufacture of glycoprotein pharmaceuticals.