Protein hydrogen exchange studied by the fragment separation method

Abstract

The potential of hydrogen-exchange studies for providing detailed information on protein structure and structural dynamics has not yet been realized, largely because of the continuing inability to correlate measured exchange behavior with the parts of a protein that generate that behavior. J. Rosa and F. M. Richards (1979, J. Mol. Biol. 133, 399-416) pioneered a promising approach to this problem in which tritium label at exchangeable proton sites can be located by fragmenting the protein, separating the fragments, and measuring the label carried by each fragment. However, severe losses of tritium label during the fragment separation steps have so far rendered the results ambiguous. This paper describes methods that minimize losses of tritium label during the fragment separation steps and correct for losses that do occur so that the label can be unambiguously located and even quantified. Steps that promote adequate fragment isolation are also described.

Fig. 1

Analysis of hemoglobin by the fragment separation method. Hemoglobin was fully tritiated at exchangeable proton sites, placed into slow exchange conditions, separated into subunits, fragmented with pepsin, and resolved by HPLC. The solid line (absorbance at 230 nm) traces elution profiles for the α- and β-chain fragments. The histogram indicates tritium count level in each fraction taken. Numbers above each peak give the fragment identity and number of protons measured/number of protons present. The value for number of protons present counts 1 for each peptide NH and zero for prolines and the N-terminal residue. Side chain Asn and Gln NH have a lifetime less than 1 min under these conditions (24) and are not expected to be recovered.

Fig. 2

Accelerated analysis of the fragment β130–146 by the fragment separation method. Fully tritiated hemoglobin, without prior separation into subunits, was fragmented with pepsin and run on an HPLC gradient designed to deliver fragment β130–146 directly. Profiles show the elution trace measured by absorbance at 230 nm and the carried tritium (histogram).

Fig. 3

Dependence of amide NH-exchange rates on pH and organic solvents. The solid line in panel A shows exchange rates of random-chain poly-DL-alanine in water at 0°C (22). The shadowed region indicates the range of rates experienced by peptide groups with various polar side chains (24). The shift in HX rate due to various mixed HPLC solvents is shown in panel B, measured using acetamide at 25°C.

Fig. 4

HX slowing factors in HPLC solvents. Slowing factors are from direct HX measurements like those in Fig. 3b, using poly-DL-alanine in mixtures of solutions A and B (see text) preset to pH 2.7. Slowing factors were measured at 25°C (●) and 13.5°C (○).

Fig. 5

Tritium loss curve for fragment β130–146 (semilog). The predicted HX curve was computed from Eq. [2] for poly-DL-alanine at pH 2.7 and 0°C taking into account the effects of Molday et al. (24). From this curve one can obtain the loss correction factor (L) for this fragment if the equivalent exchange-out time is known.

Fig. 6

A test of the HX rate prediction. The solid line is the ¹H–³H exchange curve predicted from Eq. [2] modified by the Molday et al. (24) factors for (random coil) human hemoglobin at pH 2.7 and 0°C. The data points are from ¹H–³H exchange measurements on hemoglobin at these conditions.

Fig. 7

Molar absorbance of aromatic amino acids. Extinction coefficients are shown for tyrosine and tryptophan in HPLC solvents at wavelengths pertinent for the HPLC analysis. These residues when present in a peptide fragment make a dominant contribution to measured absorbance in the HPLC elution profiles.

Fig. 8

Kinetic specificity in the proteolysis of hemoglobin by pepsin. About 12 sensitive sequences per chain are cleaved in a rapid kinetic phase that precedes the subsequent slower breakage at many sites.