Kissing bugs are one-half to one inch in length and feed on blood. Reaction to a bite can range from mild itching and swelling to life-threatening cases of anaphylactic shock. The bug can also pass on the parasite that causes Chagas' Disease.

Some components of blood-sucking insects' saliva are: prostaglandins (potent skin vasodilators); peptides that are inhibitors of enzymes in the blood-clotting cascade; molecules that prevent platelet aggregation; and molecules that cause vasodilation - blood vessel expansion. A mechanism unique to the Rhodnius prolixus blocks histamine from causing inflammation that starts the immune response. Studies of the chemistry of the substances that the insect releases from its saliva are helping to explain how R. prolixus counteracts a victim's defenses against a bug bite. The salivary glands of the insect are a cherry red color due to a heme protein - a protein with an iron-based complex. However, this heme protein does not come from the insect's blood diet. Human red blood cells contain hemoglobin - an iron-containing, oxygen-carrying molecule, whereas the heme protein in the kissing bug is an iron-containing molecule that instead carries nitric oxide (NO). Biological cells synthesize NO for specific purposes, and NO is a very important molecule for dilating blood vessels. In fact, that function is the basis of all nitric oxide donor drugs as well as other drugs that are used in humans to dilate blood vessels after a heart attack, or to lower blood pressure in hopes of preventing a heart attack. The importance of understanding the chemistry of NO and the ways in which it may be stabilized in biological systems is underscored by the 1998 Nobel Prize in medicine, given to Furchgott, Ignarro and Murad for their discoveries concerning NO as a signaling molecule in the cardiovascular system. One problem in the use of NO in living systems is that it is a very unstable molecule. It is destroyed within a minute if it is exposed to oxygen. "R. prolixus is very smart," says Walker. "It makes a protein that can bind the NO very tightly to the iron of the heme." This new class of heme proteins that carry NO is called the nitrophorins (NO-carriers). Nitrophorins are unique NO storage and transport proteins that are able to hold and keep NO stable in the insect saliva until released into the victim's tissues. Montfort's and Walker's groups have recently shown that nitrophorins in R. prolixus are exquisitely designed to store NO for long periods of time, possibly as long as a month.

Before sucking blood, the insect spits into a victim's tissues to release the nitrophorins. The dilution causes the NO to dissociate, whereby the NO enters tissues to begin dilation of the blood vessels. Montfort, Walker, and their coworkers discovered that as the NO dissociates from the heme, histamine can bind to the heme. In fact, histamine has a larger binding constant than NO. When the heme releases NO it soaks up the histamine so that there is no swelling when the kissing bug bites its victim. Thus the victim is not aware that he is being bitten. The insect gets its blood meal before the tissue puts out more histamine that causes the desire to scratch. Two functions of these nitrophorin proteins involve the release of NO to dilate the blood vessels and prevent platelet aggregation. The third function involves the uptake of histamine to delay swelling and the immune response. Montfort, Walker, and their coworkers are characterizing these nitrophorins to develop a detailed understanding of their functions. Fortunately, the nitrophorin genes have been cloned and sequenced, allowing the scientists to express the nitrophorin proteins in bacterial hosts and to obtain large amounts of protein for study. As a postdoctoral associate, John Andersen was involved in developing the expression, isolation, purification, and reconstitution protocols for the cloned proteins. Andersen is now a CoS research assistant professor of biochemistry working in Montfort's laboratory. Montfort's and Walker's groups work together to interpret the data that both groups obtain to analyze the functional and structural features of the nitrophorins. Their studies to characterize the proteins utilize X-ray crystallography, laser-flash and stopped-flow photometry, nuclear magnetic resonance (NMR), infrared spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and electrochemical techniques. Montfort is a protein crystallographer, and his group has learned how to crystallize the nitrophorin proteins and obtain their three-dimensional structures. They have also investigated the kinetics of NO binding and release. Walker's group works on the spectroscopic and electrochemical analyses. NMR is used to study dynamics - chemical changes - by analyzing the signals from protons and other nuclei in the protein macromolecule. This technology tells scientists about protein folding and unfolding, to determine the protein's flexibility. In contrast, the crystal structure of a protein shows a static picture. Both pictures are needed to completely understand how the protein functions. If there is at least one unpaired electron on the iron in the protein, then EPR can be used for analytical studies. EPR gives a particular kind of spectrum that is related to the spin-state. (In the absence of NO, the nitrophorin has 5 unpaired electrons). When the nitrophorin protein binds NO one more unpaired electron is added, giving a total of six. These six electrons pair and a signal is no longer detected. Electrochemical studies told the researchers that their nitrophorin proteins want to be in the ferric - Fe(III) - state. Unlike myoglobin in the muscles, which binds oxygen and is active in the ferrous - Fe(II) - state, nitrophorin cannot bind oxygen. In addition, NO binding in globins in the ferrous state is essentially irreversible. The unusual reversibility of NO binding to the nitrophorin heme requires a property not found in the globins - a desire to remain in the ferric state. NO is stored in a stable manner for long periods of time as a ferric nitrophorin-NO complex at the pH of the insect's saliva. NO binding to the nitrophorin is also reversible. When the pH is raised - when the nitrophorin is injected into the tissues of the victim - the protein is simultaneously diluted and NO dissociates. NO then diffuses freely through the tissues, reaching nearby capillaries where it causes dilation and brings more blood to the site of the wound. Future work will answer other fundamental questions concerning nitrophorins. The research groups know from the crystal structures that the nitrophorins have negative charges all around the NO-binding 'pocket' in the protein, and those charges stabilize the positively charged, oxidized (ferric) form of the protein. "Are all of them important," asks Walker, "or is one particular charged group most important?" To study the relative importance of the individual negative charges, the researchers are using a technique called site-directed mutagenesis. This molecular biology technique allows researchers to make specific changes in the cloned gene. The changes produce a protein that has a specific desired alteration. In this manner, various nitrophorin proteins can be created that do not have one or another of the negative charges. The structural and functional effects of the elimination of each negative charge can then be evaluated. "With site-directed mutagenesis we expect to find out which amino acid residues in the protein are really important for producing the most stable or least stable NO complexes," explains Montfort. Also intriguing is the completely unique structure of nitrophorin. All of the globins - heme binding, oxygen-carrying proteins - are alpha-helical. As an example, the form that a ribbon has when wrapped in a spiral around a pole is an alpha helix. Globins are proteins that use a number of alpha helices to create the pocket-like structure that holds the heme group and its oxygen molecule. The nitrophorin structure is unique since it is nearly all beta-barrel, and this type of structure has not been seen before for a heme protein. In contrast to other known heme proteins, nitrophorins create a beta barrel structure to form the 'pocket' that carries the heme group. Montfort's and Walker's groups conclude that nitrophorins are evolutionarily distinct from the globins - the only other heme-based gas transport proteins known. Also, nitrophorins are distinct from all other known heme protein structures. However, the beta-barrel structure is seen in another family of proteins called the lipocalins. These proteins are involved in various biological functions related to the protein's ability to bind small, hydrophobic (water-repelling) molecules within its 'pocket' inside of the barrel. The nitrophorins are also unique among lipocalins, in that the hydrophobic molecule they bind is heme. Studies of metalloproteins have established collaborations with many researchers in the CoS, as well as researchers from other UA colleges and around the world. Montfort, Walker, and M.C. Ribeiro began their collaboration with a joint grant proposal from the National Institutes of Health in 1995. Soon after, Ribeiro moved to the National Institute of Allergy and Infectious Diseases, where he is concentrating on the diseases carried by Rhodnius and other blood-sucking insects. Since then, Montfort and Walker have continued characterizing the nitrophorins, now with support from independent research grants.