Binding of ions and hydrophobic probes to alpha-lactalbumin and kappa-casein as determined by analytical affinity chromatography

The technique of analytical affinity chromatography was extended to characterize binding of ions and hydrophobic probes to proteins. Using the immobilized protein mode of chromatography, alpha-lactalbumin and kappa-casein were covalently attached to 200-nm-pore-diameter controlled-pore glass beads and accommodated for high-performance liquid chromatography. The existence of a high affinity binding site (Kdiss = 0.16 microM) (site I) for calcium ion in alpha-lactalbumin was confirmed by chromatography of [45Ca2+]. In addition, chromatography of the hydrophobic probes, 1-(phenylamino)-8-naphthalene-sulfonate (ANS)2 and 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate (bis-ANS) indicated that Ca2+ bound to a second site (presumably the zinc site or site II) with weaker affinity. Dissociation constants obtained for apo-alpha-lactalbumin were about 80 microM for ANS and 4.7 microM for bis-ANS in the absence of sodium ion. Addition of Ca2+ initially caused a reduction in surface hydrophobicity (lowered affinity for the probe dyes) followed by an increase at higher Ca2+ concentrations (greater than 0.5 mM), suggesting that occupancy of site II restores an apo-like conformation to the protein. Moreover, the effect of Zn2+ was similar to that observed in the higher Ca2+ concentration range, whereas Na+ apparently bound to site I. A calcium binding site of moderate affinity also exists in kappa-casein (Kdiss = 15.6 microM). A cluster of negative charges, probably including the orthophosphate group, most likely comprise this binding site. By preventing self-association, analytical affinity chromatography permits microscale characterization of ligand equilibria in proteins that are unaffected by protein-protein interactions.     

Characteristics of the interaction of calcium with casein submicelles as determined by analytical affinity chromatography

Interaction of calcium with casein submicelles was investigated in CaCl2 and calcium phosphate buffers and with synthetic milk salt solutions using the technique of analytical affinity chromatography. Micelles that had been prepared by size exclusion chromatography with glycerolpropyl controlled-pore glass from fresh raw skim milk that had never been cooled, were dialyzed at room temperature against calcium-free imidazole buffer, pH 6.7. Resulting submicelles were covalently immobilized on succinamidopropyl controlled-pore glass (300-nm pore size). Using 45Ca to monitor the elution retardation, the affinity of free Ca2+ and calcium salt species was determined at temperatures of 20 to 40 degrees C and pH 6.0 to 7.5. Increasing the pH in this range or increasing the temperature strengthened the binding of calcium to submicelles, similar to previous observations with individual caseins. However, the enthalpy change obtained from the temperature dependence was considerably greater than that reported for alpha s1- and beta-caseins. Furthermore, the elution profiles for 45Ca in milk salt solutions were decidedly different from those in CaCl2 or calcium phosphate buffers and the affinities were also greater. For example, at pH 6.7 and 30 degrees C the average dissociation constant for the submicelle-calcium complex is 0.074 mM for CaCl2 and calcium phosphate buffers, vs 0.016 mM for the milk salt solution. The asymmetric frontal boundaries and higher average affinities observed with milk salts may be due to binding of calcium salts with greater affinity in addition to the binding of free Ca2+ in these solutions.     

Dynamics of Male–Female Multimodal Signaling Behavior across the Estrous Cycle in Giant Pandas (Ailuropoda melanoleuca)

Giant panda courtship behavior includes multimodal signaling assemblages consisting of olfactory, vocal, and postural elements. While signaling is generally conspicuous, successful copulation is inconsistently achieved in captivity, even when female behavioral and physiological data indicate that ovulation is imminent. We set out to characterize these complex patterns of social behavior by observing interactions between 26 unique pairs of giant pandas housed in adjoining pens throughout the females' reproductive cycle. We categorized social behaviors from a transactional perspective and examined social exchanges via analyses of the relative frequency of social behaviors, and via the sequential relationship between male and female social behavior. From non‐estrus to peak‐estrus, we found that the relative frequency of female affiliative and sexual behavior increased and that the relative frequency of ambivalent and aggressive behavior decreased. Male behavior was fairly constant, except for sexual behavior, which increased during peak‐estrus, when it was facilitated by female sexual behavior. Sequential analysis of social interactions showed that preceding behavior had a significant influence over the other panda's response behavior primarily during peak‐estrus, suggesting that pandas are most responsive to conspecific signaling during the peri‐ovulatory period. However, behavioral momentum was a dominant feature of the intra‐individual transitions. Females maintained sexual, ambivalent, and neutral behavior during interactions significantly more than would be expected by chance, with male behavior bearing little influence once the behavior was initiated. A similar pattern was also observed in males, who maintained affiliative, interested, and neutral behaviors. Overall, our data suggest that the multimodal signals used by giant pandas during courtship do not consistently evoke a discrete, immediate response from receivers. Instead, signals appear to advertise reproductive condition and influence potential mates over longer timeframes, suggesting the potential tonic role of communication.     

Purification and characterization of the fusion protein trypsin-streptavidin expressed in Escherichia coli

Expression of fusion protein trypsin-streptavidin (TRYPSA)⁴ in Escherichia coli was evaluated and the protein purified. Protein expression was induced by 1 mM isopropylthio--D-galactoside (IPTG), and the enzyme activity was measured by the hydrolysis rate of p-toluenesulfonyl-l-arginine methyl ester (TAME). Expression of the fusion protein in the cell-free extract decreased with increased induction time; correspondingly, that in the inclusion bodies increased. The total expression in Luria–Bertani broth (LB) and Terrific Broth (TB) media reached the highest levels in 2 hr at 30°C. The optimum expression level was 35 and 48 U/L in LB and TB, respectively. Expression of the fusion protein was verified by Western Blot analysis using streptavidin antiserum, and the fusion protein was purified using a benzamidine Sepharose 6B affinity column at room temperature. The molecular size of the soluble purified fusion protein was determined by size-exclusion chromatography using Superose 12 FPLC. A molecular weight of 39–40 kDa was obtained, indicating that the soluble protein exists as a monomer; thus, the presence of the trypsin domain must prevent the streptavidin domain from tetramer formation.     

Development of a Streptavidin-Conjugated Single-Chain Antibody That Binds Bacillus cereus Spores

Control of microorganisms such as Bacillus cereus spores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encode B. cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of B. cereus spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 × 10⁴ B. cereus spores per ml. Greater than 90% of the B. cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.     

Effects of season and social interaction on fecal testosterone metabolites in wild male giant pandas: implications for energetics and mating strategies

In the first-ever study of reproductive endocrinology in wild male giant pandas (Ailuropoda melanoleuca), we provide new insights into the reproductive ecology of the species. We tracked and observed pandas in Foping Nature Reserve of the Qinling Mountains for 3 years, collecting fecal samples for testosterone metabolite analysis and data on reproductive activity. Males encountered multiple potential mates and competed for reproductive access to females. Male testosterone metabolites increased in February, peaked in March and April, and fell back to baseline after the mating season. However, males did not maintain a high testosterone level throughout the mating season. Male testosterone instead peaked during encounters with potential mates and declined between encounters. These results indicate that testicular activity is typically dormant until mobilized by interactions with females and potentially by interactions with male competitors. This suggests that male pandas may be energetically constrained, elevating testosterone levels only when necessary to meet the demands of intrasexual competition and courtship and fertilization of females. Maintaining a high testosterone level is metabolically expensive and male pandas enter the mating season during a period of low food availability. If this hypothesis is correct, male panda body condition may be an important determinant of reproductive outcome, and anthropogenic activities that diminish foraging resources may have significant impacts on the mating ecology of the species, affecting its conservation.     

Hormonal and behavioral relationships during estrus in the giant panda

Nonlactating female giant pandas experience a single estrus during the spring mating season. Although both hormonal and behavioral aspects of estrus have been reported on captive females, little is known about the relationship between them. Data were collected on a single captive female during three successive seasons, revealing a high degree of regularity in both estrogen profiles and the temporal pattern of associated behaviors. The birth of a live cub in the last of the three seasons, using artificial insemination, confirmed the time of ovulation as occurring shortly after a peak in urinary estrogen values. In this study, we examined the occurrence of female scent marking, rear presentations, chirp and bleat vocalizations, and the tail‐up display, and plotted their occurrence relative to the underlying estrogen values. We conclude that the role of estrogen in the expression of these behaviors is neither simple nor directly causal. Temporal associations indicate that a triggering rather than a maintenance function for estrogen is implicated. This analysis provides a clear identification of estrous behaviors, but in both the time and form of occurrence these behaviors most likely vary between females and seasons. Zoo Biol 20:537–543, 2001. © 2002 Wiley‐Liss, Inc.