Quenching of room temperature protein phosphorescence by added small molecules.

A number of molecular agents that can efficiently quench the room temperature phosphorescence of tryptophan were identified, and their ability to quench the phosphorescence lifetime of tryptophan in nine proteins was examined. For all quenchers, the quenching efficiency generally follows the same sequence, namely, N-acetyltryptophanamide (NATA) greater than parvalbumin approximately lactoglobulin approximately ribonuclease T1 greater than liver alcohol dehydrogenase greater than aldolase greater than Pronase approximately edestin greater than azurin greater than alkaline phosphatase. Quenching rate constants for O2 and CO are relatively insensitive to protein differences, while H2S and CS2 are somewhat more sensitive. These small molecule agents appear to act by penetrating into the proteins. However, penetration to truly buried tryptophans is less favorable than previously suggested; in five proteins studied, quenching efficiency by O2 is 20-1000 times lower than for NATA, and up to 10(5) lower for H2S and CS2. Larger and more polar quenchers--including organic thiols, conjugated ketones and amides, and anionic species--were also studied. The efficiency of these quenchers does not correlate with quencher size or polarity, the quenching reaction has low energy of activation, and quenching rates are insensitive to solvent viscosity. These results indicate that the larger quenchers do not approach the buried tryptophans by penetrating into the proteins, even on the long phosphorescence time scale, and are also inconsistent with a mechanism in which quencher encounter with the tryptophan occurs in free solution, as in a protein-opening reaction. The results obtained suggest that the quenching process involves a long-range radiationless transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

On the prevalence of room-temperature protein phosphorescence.

A large number of proteins were tested for the property of intrinsic phosphorescence in deoxygenated aqueous solution at room temperature. The majority of proteins exhibit phosphorescence under normal solution conditions. Phosphorescence lifetimes from 0.5 millisecond to 2 seconds were observed in three-fourths of the proteins tested. The lifetime appears to correlate with relative isolation of the tryptophan indole side chain from solvent. With few exceptions, proteins in general can be expected to display a phosphorescence lifetime greater than 30 microseconds. This widespread characteristic of proteins has been largely overlooked because long-lived phosphorescence is highly sensitive to quenching by low levels of dissolved oxygen in solution. Protein phosphorescence offers a new time domain and a far wider dynamic range than has been used before for photoluminescence experimentation.

Biochemistry without oxygen.

Published procedures for experimentation under anoxic conditions generally involve specialized apparatus that hinders the easy manipulation of experimental samples. We describe here some procedures that rapidly remove oxygen from experimental solutions, maintain anoxia with simple equipment for long periods of time, and do not interfere with normal sample addition and removal, spectrometric measurements, chromatographic manipulations, and the like. Anoxia can be achieved and maintained by the use of an enzyme system (glucose oxidase, glucose, catalase), or an inorganic oxygen-reducing system (ferrous pyrophosphate), or dithionite. Physical isolation of experimental samples from atmospheric oxygen can be maintained by continuous flushing with treated argon gas and/or by an overlay of heavy mineral oil.

Protein fluorescence quenching by small molecules: protein penetration versus solvent exposure.

Experiments were done to test the thesis that acrylamide and similar small molecules can penetrate into proteins on a nanosecond time scale. The approach taken was to measure the pattern of fluorescence quenching exhibited by quenching molecules differing in molecular character (size, polarity, charge) when these are directed against protein tryptophans that cover the whole range of tryptophan accessibility. If quenching involves protein penetration and internal quencher migration, one expects that larger quenchers and more polar quenchers should display lesser quenching. In fact, no significant dependence on quencher character was found. For proteins that display measurable quenching, the disparate quenchers studied display very similar quenching rate constants when directed against any particular protein tryptophan. For several proteins having tryptophans known to be buried, no quenching occurs. These results are not consistent with the view that the kinds of small molecules studied can quite generally penetrate into and diffuse about within proteins at near-diffusion-limited rates. Rather the results suggest that when quenching is observed, the pathway involves encounters with tryptophans that are partially exposed at the protein surface. Available crystallographic results support this conclusion.

Internal protein motions, concentrated glycerol, and hydrogen exchange studied in myoglobin.

Experiments were carried out to measure the effect of concentrations of glycerol on H-exchange (HX) rates by using myoglobin as a test protein. Concentrated glycerol has only a small slowing effect on the HX kinetics of freely exposed amides, studied in a small molecule model (acetamide). Larger effects occur in structured proteins. The effect of solvent glycerol on different parts of the HX curve of myoglobin was studied by use of a selective "kinetic labeling" approach. Concentrated glycerol exerts an apparently reverse effect on protein H exchange; the faster exchanging "surface" protons are least affected, while the slower and slower amide NH is further slowed by larger and larger factors. These results seem inconsistent with solvent penetration models which generally visualize slower and slower protons as being placed, and undergoing exchange, farther and farther from the solvent-protein interface. On the other hand, the results are as expected for the local unfolding model for protein H exchange since concentrated glycerol is known to stabilize proteins against unfolding. In the local unfolding model, slower exchanging protons are released by way of higher energy and therefore generally larger, unfolding reactions. Larger unfoldings must be more inhibited by the glycerol effect.

Penetration of dioxygen into proteins studied by quenching of phosphorescence and fluorescence.

Experiments were done to measure the ability of dioxygen to collisionally quench the phosphorescent and fluorescent tryptophans in alcohol dehydrogenase and alkaline phosphatase. In all cases, luminescence is quenched with rate constants close to 1 x 10(9) M-1 s-1. The rate of reaching the buried tryptophans is little affected by solvent viscosity due to added glycerol. Quenching by dioxygen is not due to a protein-opening reaction. It appears to be rate limited by internal protein diffusion rather than at the entry step. Dioxygen appears to enter the proteins directly, as in liquidlike diffusion, rather than through transiently forming channels that are only present a small fraction of the time. A high-pressure oxygen system is described that considerably facilitates fluorescence quenching experiments.

A high energy structure change in hemoglobin studied by difference hydrogen exchange.

The hydrogen exchange behavior of a small allosterically responsive set of exchanging hydrogens was studied in hemoglobin A and in some chemically modified hemoglobins. The set experiences an exceptionally large change in exchange rate through hemoglobin's allosteric transition. This indicates, according to the local unfolding model of H-exchange, that a large change in allosteric free energy impinges on the opening segment that exposes these protons to exchange. In oxyhemoglobin the set consists of 5 to 6 protons which exchange with a half-time of 20 s at pH 7.4 and 0 degrees C. In deoxyhemoglobin the set splits into a slower and a faster half. The slower 3 protons exchange more slowly than in oxyhemoglobin by a factor of 5000 (26 h half-time) and are 5-fold slower still in the presence of pyrophosphate or inositol hexaphosphate (136 h half-time). The other 2 to 3 protons exchange about 20-fold faster in both cases (about 2 h and 10 h half-times). The effect of some chemical modifications was tested, including reaction with iodoacetamide and N-ethylmaleimide and cleavage with carboxypeptidases A and B. In all cases the 3 slower protons continue to behave as a cohesive set and in the various modified deoxyhemoglobins their exchange is accelerated by factors ranging between 1 and 3 decades. These factors correlate with the effect of the different modifications on hemoglobin cooperativity.

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.