Allan L. Bieber

Mass spectrometric immunoassay.

A new, general method of immunoassay is demonstrated. The approach is based on the microscale immunoaffinity capture of target antigens followed by mass-specific identification and quantitation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Immunoaffinity capture of antigens effectively overcomes signal suppression effects typically encountered during traditional matrix-assisted laser desorption/ionization analysis of complex biological mixtures while simultaneously concentrating the analyte into a small volume. Mass spectrometric detection of antigens is unambiguous, as antigen signals are observed at characteristic mass-to-charge values in the mass spectrum, offering a high level of immunity to artifacts due to nonbiospecific retention of mixture components. However, the most important aspect of such mass-specific detection is the ability to use a single assay to screen biological systems for the presence of multiple, mass-resolved antigens. Analyte quantitation is possible by using a single antibody to capture both the antigen and an antigen variant which has been chemically modified to have a different mass. With proper calibration, the relative signal intensities of the two species in the mass spectrum can be used to determine the antigen concentration. Sample incubation and processing methods were such that a typical analysis could be performed in less than 1 h while subnanomolar sensitivities were maintained. The technique has been used for the rapid, selective, and quantitative screening of human blood for the presence of myotoxin a, and Mojave toxin form the venoms of the prairie rattlesnakes, Crotalus viridis viridis, and and the Mojave rattlesnake, Crotalus scutulatus scutulatus.

Allurin, a 21-kDa sperm chemoattractant from Xenopus egg jelly, is related to mammalian sperm-binding proteins

Previously, we demonstrated that a protein from Xenopus egg jelly exhibits sperm chemoattractant activity when assayed by either video microscopy or by sperm passage across a porous filter. Here we describe the isolation and purification of allurin, the protein responsible for this activity. Freshly oviposited jellied eggs were soaked in buffer, and the conditioned medium was loaded onto an anion exchange column and eluted with an NaCl gradient. The active fraction was purified further by RP-HPLC, the chemoattractant protein appearing as a single sharp peak. The amino acid sequence of the protein, determined by direct sequencing and cloning of cDNAs coding for the protein, consisted of 184 amino acids having a molecular mass of 21,073 Da. The protein shares homology with the mammalian cysteine-rich secretory protein (CRISP) family that includes testes-specific spermatocyte protein 1, a cell adhesion protein which links spermatocytes to Seritoli cells, and acidic epididymal glycoproteins that bind to sperm and have been implicated in sperm–egg fusion. Phylogenetic analysis suggests that allurin evolved from the ancestral protein that gave rise to the mammalian CRISP family. Addition of allurin to this family portends that the CRISP family represents a group of “sperm escort” proteins, which bind to sperm at various steps in their life history, facilitating passage from one functional stage to the next. Allurin stands out in this regard, representing both the first vertebrate sperm chemoattractant to be purified and sequenced and the first member of the CRISP family to be found in the female reproductive tract.

Purification and characterization of mojave (Crotalus scutulatus scutulatus) toxin and its subunits

Abstract An acidic lethal protein, Mojave toxin, has been isolated from the venom of Crotalus scutulatus scutulatus. The purified toxin had an i.v. LD50 of 0.056 μg/g in white mice. Disc polycrylamide gel electrophoresis at pH values of 9.6 and 3.8 and isoelectric focusing in polyacrylamide gels with a pH 3.5–10 Ampholyte gradient were used to establish the presence of one major protein band. The pI of the most abundant form of the toxin was determined to be 5.5 by polyacrylamide gel isoelectric focusing experiments. The molecular weight was established to be 24,310 daltons from amino acid composition data. Mojave toxin was shown to consist of two subunits, one acidic and one basic with isoelectric point (pI) values of 3.6 and 9.6, respectively. Amino acid analyses established molecular weights of 9593 for the acidic component and 14,673 for the basic component. The acidic subunit consisted of three peptide chains intermolecularly linked by cystine residues. The basic subunit was a single polypeptide chain with six intramolecular disulfide bonds. The basic subunit was lethal to test animals with an intravenous LD50 of 0.58 μg/g. Following recombination of the subunits a recombinant toxin was isolated which was identical to the native toxin by comparisons of electrophoretic mobility and toxicities. Comparisons of circular dichroism spectra also indicated reassociation to the native toxin structure. Phospholytic activity was associated with Mojave toxin and the basic subunit was responsible for this enzymic activity. Phospholipase activity of the basic subunit was inhibited by addition of the acidic subunit.

Fractionation of midget faded rattlesnake (Crotalus viridis concolor) venom: lethal fractions and enzymatic activities.

Abstract The venom of the midget faded rattlesnake has been resolved into eight fractions using anion-exchange chromatography. Fractions 1, 6 and 7 were lethal to mice at protein concentrations equivalent to 3 × ld 50 of the crude venom. Fraction 7 ( ld 50 , 0·12 μg/g) contained the major lethal component(s) of the venom. Using disc gel electrophoresis only one major and one minor protein component were present in this fraction. One major and one minor component, both with pI values less than 5, were also observed after isoelectric focusing. One protein band with a pI > 9 was present in Fraction 1 after isoelectric focusing. Injection of this fraction resulted in paralysis of the hind limbs and respiratory distress immediately after injection. Crude venom and each fraction were tested for phosphodiesterase, phospholipase, l -amino acid oxidase, arginine ester hydrolase and phosphomonoesterase activities. Phosphodiesterase activity was detected only in Fraction 1. Phospholipase activities were present in Fractions 6 and 7. l -amino acid oxidase activity was present in Fractions 1 and 2. Arginine ester hydrolase activity was most prominent in Fractions 2–5. Phosphomonoesterase activity was not detectable in crude venom nor in any fraction derived therefrom.

Antigenic relationships between Mojave toxin subunits, Mojave toxin and some crotalid venoms.

Immunochemical responses of a number of pit viper venoms to antibodies derived separately from the acidic and basic subunits were investigated by enzyme linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion. The polyclonal antisera to the basic subunit were generated in rabbits, whereas mouse hybridoma cell cultures were used to produce antibodies to the acidic subunit. The immunochemical response of a venom correlated well with published values for LD50 dose for the test venom. Many venoms that elicited a positive response with antiserum to the basic subunit also reacted strongly with the hybridoma derived antibodies to the acidic subunit. The data support the conclusion that crotalid venoms which are more lethal have in common a potent venom component that is immunochemically related to Mojave toxin.

The complete sequence of the acidic subunit from Mojave toxin determined by Edman degradation and mass spectrometry.

Mojave toxin, a heterodimeric, neurotoxic phospholipase complex from Crotalus scutulatus scutulatus, is one of a group of closely related rattlesnake toxins for which much structural information is still lacking. The complete amino-acid sequence of the acidic subunit from Mojave toxin was determined. The three individual peptide chains, derived from the acidic subunit by reductive alkylation, were separated by high-performance liquid chromatography. Fragmentations of the A and B chains were done using specific proteinases and the resulting peptide mixtures were fractionated by reverse-phase high-performance liquid chromatography. Sequence analyses on the intact chains and the fragments from digests were done by automated Edman degradation, carboxypeptidase Y degradation and triple-quadrupole and tandem-quadrupole Fourier-transform mass spectrometry. The sequence for each acidic subunit chain is very similar to the corresponding chain from the related neurotoxin complex, crotoxin, and overall the sequence is similar to the sequences of group I and II phospholipases A2. The N-terminus of the B chain is blocked by pyroglutamic acid. The existence of two distinct and closely related C chains was established. It is unlikely that the small sequence difference can account for the isoforms that are present in purified Mojave toxin and in unfractionated venom.

Enzymatic activation of the Bacillus sphaericus mosquito larvicidal toxin

Bacillus sphaericus insecticidal toxin was activated by trypsin, α-chymotrypsin, and mosquito gut homogenates to a form which was cytotoxic to cultured mosquito cells. Gut extracts from highly resistant Aedes aegypti larvae were as effective in activating this toxin as extracts from the highly sensitive species Culex quinquefasciatus. Activation altered the apparent molecular weight of the toxin by ca. 2–4 kDa.

Amino acid sequences of myotoxins from Crotalus viridis concolor venom

Myotoxins I and II were isolated from the venom of Crotalus viridis concolor. Complete sequences were derived for each reduced, alkylated toxin with data obtained by a single run on a gas phase sequencer and from fragments derived by cyanogen bromide cleavage. The results demonstrate that microheterogeneity is present in myotoxin II. The newly established sequences were compared with 3447 protein sequences in the Protein Information Resource database. The only homologous proteins found were other known myotoxins from rattlesnake venoms, namely myotoxin a, crotamine and peptide C.

Structural, biological and biochemical studies of myotoxin a and homologous myotoxins

AbstractMyotoxin a and a group of closely related, small, basic toxins cause myonecrotic destruction of muscle tissue upon envenomation. The sarcoplasmic reticulum swells and eventually breaks down to small vesicles. Degeneration of myofibrils and myofilaments ensues and loss of the classic striation pattern is apparent. These toxins exhibit high sequence similarity as well as sequence microheterogeneity. A conformational heterogeneity was recently discovered in myotoxin a. The existence of myotoxin a in two forms in equilibrium in solution hinders the generation of a well defined three-dimensional structure. The difference, if any, in the biological activity of the two forms has not been established yet. Recent biochemical studies indicate that myotoxin a is a potent Ca2+ releasing agent that binds to calsequestrin in the lumen of the sarcoplasmic reticulum.

Crisp proteins and sperm chemotaxis: discovery in amphibians and explorations in mammals

Crisp proteins appear to play multiple roles in the life history of sperm. One of these roles is to act as a sperm chemoattractant. Allurin, a 21 kDa Crisp protein rapidly released from the egg jelly of at least two frogs, X. laevis and X. tropicalis, elicits directed motility in both homospecific and heterospecific sperm. In X. tropicalis, allurin is coded for by the newly documented Crisp A gene. Recently, the observation that allurin can also elicit chemotaxis in mouse sperm raises the question of whether allurin-like proteins might act as sperm chemoattractants in mammals. Although an allurin gene has yet to be documented in mammals, Crisp proteins truncated post-translationally appear to exist in both the male and female reproductive tract of mammals.