Biomedical subjects

J R Rogero

Publications and source records attributed to J R Rogero.

22 records · Page 2Linked to original sources

PubMed1985

Protein hydrogen exchange studied by the fragment separation method.

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.

Chemical Phenomena↗

PubMed1984

Crotoxin. I. Immunology and interaction of the subunits.

p6ospholipase A and crotapotin were purified by gel filtration and ion-exchange chromatography from Crotalus durissus terrificus venom. The minimal molecular weights obtained by amino acid analysis were 13,400 daltons for phospholipase A and 8,300 daltons for crotapotin. Phospholipase A and crotapotin form a 1:1 molar complex that was stable during gel filtration. The LD50 of phospholipase A was increased about 10-fold when in a 1:1 complex with crotapotin. Crotapotin, phospholipase A and crotoxin (crotapotin + phospholipase A) gave precipitin reactions when tested with specific rabbit antisera. Immunological studies of these proteins using crotalus antiserum confirmed 1:1 molar ratio of the complex. Crotoxin antiserum also reacts with either crotapotin or phospholipase A separately. Similar antigenic determinants were observed in the subunits and it seems that some antigenic determinants are masked in the phospholipase A-crotapotin complex.

Antigens↗

PubMed1983

Identification of an allosterically sensitive unfolding unit in hemoglobin.

Hydrogen-exchange studies locate a set of seven allosterically sensitive amide NH protons side by side around two turns of the F-FG helical segment in the hemoglobin beta chain. Some of these protons are on the aqueous protein surface and some deeply inside, yet they all exchange with solvent protons at similar rates. Further, they move in unison to a new common rate when hemoglobin changes its allosteric form. These observations and analogous results for other proteins appear to be inconsistent with penetration-dependent models which relate H-exchange rate to solvent accessibility in the native state. Rather, these results point to sizeable fluctuational distortions that make small sets of protons more or less equally accessible in some transient H-exchange transition state, as visualized in the local unfolding model. The set of allosterically sensitive protons studied here exchanges 30-fold faster in liganded hemoglobin than in the deoxy form. In terms of the unfolding model, this means that the F-FG structure is relatively destabilized in oxyhemoglobin, so that the allosterically linked change in structural free energy at F-FG favors the deoxy state. The 30-fold change in H-exchange rate suggests a contribution to the allosteric free energy by this segment of 2 kcal (1 cal = 4.184 J). These experiments utilized a labeling technique, described earlier, that selectively places tritium on sites whose H-exchange rates are sensitive to the protein functional state, and used a method introduced by Rosa & Richards (1979,1981) to locate this label in the protein. The latter method, which rapidly separates protein fragments under conditions that can preserve exchangeable label, was here brought to a more quantitative level. Taken together, these techniques provide a "functional labeling" method capable of selectively labeling and identifying protein segments that participate in functional interactions.

Allosteric Site↗

PubMed1980

Individual breathing reactions measured in hemoglobin by hydrogen exchange methods.

Protein hydrogen exchange is generally believed to register some aspects of internal protein dynamics, but the kind of motion at work is not clear. Experiments are being done to identify the determinants of protein hydrogen exchange and to distinguish between local unfolding and accessibility-penetration mechanisms. Results with small molecules, polynucleotides, and proteins demonstrate that solvent accessibility is by no means sufficient for fast exchange. H-exchange slowing is quite generally connected with intramolecular H-bonding, and the exchange process depends pivotally on transient H-bond cleavage. At least in alpha-helical structures, the cooperative aspect of H-bond cleavage must be expressed in local unfolding reactions. Results obtained by use of a difference hydrogen exchange method appear to provide a direct measurement of transient, cooperative, local unfolding reactions in hemoglobin. The reality of these supposed coherent breathing units is being tested by using the difference H-exchange approach to tritium label the units one at a time and then attempting to locate the tritium by fragmenting the protein, separating the fragments, and testing them for label. Early results demonstrate the feasibility of this approach.

Deuterium↗