The denaturation of trypsin

The denaturation of α- and β-trypsin in alkaline and neutral solution was studied. The denaturation of α-trypsin was a strict second-order reaction at neutrality. However, the denaturation of β-trypsin was not a pure secondorder reaction at the same pH. Calcium ion retarded the rate of β-trypsin denaturation to a greater extent than that of α-trypsin. In alkaline solution, trypsin has very short half life ($\text{t}{}_{\mathrm{<mtext>case1</mtext><mtext>2</mtext>}}\text{< 30 minutes}$). On the other hand, the denaturation of immobilized trypsin in alkaline solution is a first-order reaction as is immobilized chymotrypsin. The rates of these two denaturations are similar. Calcium ion does not affect the rate of trypsin denaturation in alkaline solution.     

Studies on alpha-amylase from a thermophilic bacterium. II. Thermal stability of the thermophilic alpha-amylase.

The effect of pH, mental ions, and denaturing reagents on the thermal stability of thermophilic alpha-amylase [EC 3.2.1.1] were examined. The enzyme was most stable at around pH 9.2, which is coincident with the isoelectric point of the enzyme. The stability of the enzyme was increased by the addition of calcium, strontium, and sodium ions. The addition of calcium ions markedly stabilized the enzyme. The protective effects of calcium and sodium ions were additive. At room temperature, no detectable destruction of the helical structure of the enzyme was observed after incubation for 1 hr in the presence of 1% sodium dodecylsulfate, 8 M urea or 6 M guanidine-HC1. The addition of 8 M urea or 6 M guanidine-HC1 lowered the thermal denaturation temperature of the enzyme. The enzyme contained one atom of tightly bound intrinsic calcium per molecule which could not be removed by electrodialysis unless the enzyme was denatured. The rate constants of inactivation and denaturation reactions in the absence and presence of calcium ions were measured and thermodynamic parameters were determined. The presence of calcium ions caused a remarkable decrease in the activation entropy.     

Sepharose-bound trypsin and activated sepharose-bound trypsinogen. Studies with small and large substrates

Several enzymic and physical properties of Sepharose-bound trypsin and activated Sepharose-bound trypsinogen have been compared to those of the soluble enzyme. Sepharose-bound trypsinogen could be activated to the same extent as soluble trypsinogen; the release of the activation peptide and formation of the active site occurred as expected in the presence of catalytic amounts of trypsin. With synthetic substrates, the relative activity and pH dependence of both immobilized trypsin preparations were essentially identical and nearly the same as the soluble enzyme. Sepharose-trypsin also formed an inactive complex with soybean trypsin inhibitor, with 85% of the active sites participating. In contrast, the activity of Sepharose-trypsin with chymotrypsinogen and with trypsinogen as substrates was only 40% that of soluble trypsin. There is evidence for some catalytic heterogeneity of active sites of bound trypsin; probably those sites buried within the gel have a limited catalytic efficiency with macromolecular substrates. The immobilized enzyme is more stable than the soluble enzyme at elevated temperatures and to concentrated urea, and denaturation by urea at pH 8 is fully reversible since the loss of molecules by autolysis is eliminated.     

Immobilization of trypsin on sand: Mode of binding

Acid-washed and heat-treated river sand was separated into different fractions by geochemical methods and immobilization of trypsin was carried out on the separated fractions using 3-aminopropyltriethoxysilane and glutaraldehyde. Scanning Electron Micrographs of the purified fraction (Sp. gr >2.5 and <2.8) of magnetically non-susceptible sand and quartz showed that the enzyme could be fixed on the supports. Malonic acid (16.3 nmol and 16.7nmol per g) appeared to be bound to alkylamine purified fraction of magnetically non-susceptible sand and alkylamine quartz, respectively. Studies on the effect of 6 M guanidine.HCl on immobilized trypsin demonstrated that immobilized trypsin had considerable stability against denaturation. The results obtained indicated that magnetically non-susceptible sand was found to be nearly as good as quartz for trypsin immobilization and that trypsin was covalently coupled to sand via 3-aminopropyltriethoxysilane and glutaraldehyde.     

The role of bound calcium ions in thermostable, proteolytic enzymes. I. Studies on thermomycolase, the thermostable protease fron the fungus Malbranchea pulchella.

Thermomycolase, the thermostable, extracellular, serine protease of the fungus Malbranchea pulchella (G. Voordouw, G. M. Gaucher, and R. S. Roche (1974), Can J. Biochem. 52, 981-990), binds one calcium ion with an apparent binding constant of 5 X 10(5) M-1 at 25degreesC, pH 7.50, and ionic strength 0.1. There is very little change in the overall conformation of thermomycolase upon binding of this calcium ion: no change can be detected, beyond experimental error, in the sedimentation coefficient or the aromatic and peptide circular dichroism of the enzyme. However, binding of calcium changes the absorption spectrum, the ultraviolet difference spectrum being characterized by a strong band at 237 nm and smaller bands at 280 and 295 nm. The difference molar extinction coefficient at 237 nm parallels the calcium-binding isotherm. The small changes in equilibrium properties are constrasted by large calcium-dependent changes in the rates of autolytic degradation and thermal and urea denaturation. The dependence of the second-order rate constant for autolytic degradation on the free calcium ion concentration can be quantitatively accounted for by a model in which only conformers with an unoccupied calcium binding site serve as substrates in the reaction. The calcium dependence of the first-order rate constant for thermal denaturation at 70degreesC and pH 7.0 can also be accounted for quantitatively by a model in which the critically activated intermediate has a smaller calcium-binding constant than the native form of the enzyme under these conditions. The same model also accounts for the denaturation in 8 M urea at 50degreesC, pH 7.0. Rates of hydrogen-tritium exchange are shown to decrease when the calcium ion is bound. Irrespective of the occupancy of the calcium-binding site 33% of the backbone peptide hydrogens of thermomycolase do not exchange within 24 hr at 25degreesC, pH 8.0, and ionic strength 0.1.     

Efficient Tandem LysC/Trypsin Digestion in Detergent Conditions

All shotgun proteomics experiments rely on efficient proteolysis steps for sensitive peptide/protein identification and quantification. Previous reports suggest that the sequential tandem LysC/trypsin digest yields higher recovery of fully tryptic peptides than single‐tryptic proteolysis. Based on the previous studies, it is assumed that the advantageous effect of tandem proteolysis requires a high sample denaturation state for the initial LysC digest. Therefore, to date, all systematic assessments of LysC/trypsin proteolysis are done in chaotropic environments such as urea. Here, sole trypsin is compared with LysC/trypsin and it is shown that tandem digestion can be carried with high efficiency in Mass Spectrometry‐compatible detergents, thereby resulting in higher quantitative yields of fully cleaved peptides. It is further demonstrated that higher cleavage efficiency of tandem digests has a positive impact on absolute protein quantification using intensity‐based absolute quantification (iBAQ) values. The results of the examination of divergent urea tandem conditions imply that beneficial effects of the initial LysC digest do not depend on the sample denaturation state, but, are mainly caused by different target specificities of LysC and trypsin. The observed detergent compatibility enables tandem digestion schemes to be implemented in efficient cellular solubilization proteomics procedures without the need for buffer exchange to chaotropic environments.     

Protein phosphorylation influences proteolytic cleavage and kinase substrate properties exemplified by analysis of in vitro phosphorylated Plasmodium falciparum glideosome-associated protein 45 by nano-ultra performance liquid chromatography-tandem mass spectrometry

Plasmodium falciparum glideosome-associated protein 45 (PfGAP45) was in vitro phosphorylated by P. falciparum calcium-dependent protein kinase (PfCDPK1) and digested using the four proteases trypsin, chymotrypsin, AspN, and elastase. Subsequently, phosphopeptide enrichment using Ga(III) immobilized metal affinity chromatography (IMAC) was performed. The resulting fractions were analyzed using ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), resulting in the identification of a total of nine phosphorylation sites: Ser31, Ser89, Ser103, Ser109, Ser121, Ser149, Ser156, Thr158, and Ser173. During in-depth analyses of the detected phosphopeptides, it was observed that phosphorylation alters the properties of PfGAP45 as kinase and protease substrate. The closely adjacent phosphorylation sites Ser156 (major site) and Thr158 (minor site) were analyzed in detail because at first glance the specific proteases gave highly variable results with respect to the relative abundance of these sites. It was observed that (i) formation of pSer156 and pThr158 was mutually exclusive and (ii) phosphorylation at Ser156 or Thr158 interfered specifically with proteolysis by chymotrypsin or trypsin, respectively. The latter effect was studied in detail using synthetic phosphopeptides carrying either pSer156 or pThr158 as substrate for chymotrypsin or trypsin, respectively.     

Immobilization of trypsin on poly(urea-formaldehyde)-coated fiberglass cores in microchip for highly efficient proteolysis

Trypsin was covalently immobilized on poly(urea‐formaldehyde)‐coated fiberglass cores based on the condensation reaction between poly(urea‐formaldehyde) and trypsin for efficient microfluidic proteolysis in this work. Prior to use, a piece of the trypsin‐immobilized fiber was inserted into the main channel of a microchip under a magnifier to form a core‐changeable bioreactor. Because trypsin was not permanently immobilized on the channel wall, the novel bioreactor was regenerable. Two standard proteins, hemoglobin (HEM) and lysozyme (LYS), were digested by the unique bioreactor to demonstrate its feasibility and performance. The interaction time between the flowing proteins and the immobilized trypsin was evaluated to be less than 10 s. The peptides in the digests were identified by MALDI‐TOF MS to obtain PMF. The results indicated that digestion performance of the microfluidic bioreactor was better than that of 12‐h in‐solution digestion.     

Inactivation and reactivation of horseradish peroxidase immobilized by various procedures.

Horseradish peroxidase (HRP) immobilized by coupling the amino acid side chain amino groups or carbohydrate spikes to the matrix has been studied for its resistance to heat, urea-induced inactivation and ability to regain activity after denaturation in order to understand the influence of the nature of immobilization procedure on these processes. The various immobilized preparations were obtained and their properties studied: Sp-HRP was obtained by direct coupling of HRP to cyanogen bromide-activated Sepharose, Sp-NHHRP by coupling periodate oxidized and diamine-treated enzyme to the cyanogen bromide activated Sepharose, SpNH-COHRP by coupling periodate-treated enzyme to amino-Sepharose and SpCon A-HRP by binding of the enzyme on Con A-Sepharose. All the immobilized preparations exhibited higher stability against heat-induced inactivation as compared to the native HRP. Sp-NHHRP was most stable followed by Sp-HRP, SpNH-COHRP and SpCon A-HRP. Sp-NHHRP was also superior in its ability to regain enzyme activity after thermal denaturation, although Sp-HRP regained maximum activity after urea denaturation. Inclusion of Ca2+ was essential for the reactivation of all preparations subsequent to denaturation by urea.     

Role of the O-phosphoserine clusters in the interaction of the bovine milk αs1-, β-, κ-caseins and the PP3 component with immobilized iron (III) ions

α_s1- and β-Caseins have a sequence cluster -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- which is not present in κ-casein and the whey PP3 component. The affinity of these phosphoproteins for the iron(III)-iminodiacetic acid (IDA) complex immobilized on Sepharose was studied a a function of pH, urea concetnration, calcium ion concentration, enzymatic dephosphorylation and temperature. The affinity of the three polyphosphorylated proteins (α_s1- and β-caseins, PP3) was similar. The sequence cluster was not a specific recognition pattern for the iron(III) ion. These three proteins presented a site of high affinity and a site of weak affinity. κ-Casein, which had only one Ser(P) residue, presented only the site of weak affinity. Their primary site which was absent after dephosphorylation or calcium ion addition required the presence of at least two Ser(P) residues close in space. Their secondary site was sensitive to the presence of urea. It was sensitive to pH variation for PP3 and κ-casein. The study of the affinity of a few free amino acids towards iron(III)-IDA showed that the secondary site involved tryptophan and tyrosine residues for α_s1- and β-caseins, histidine residues for PP3 and cysteine residues for κ-casein.     

Glass-immobilized glycated-trypsin: A novel modified trypsin that is remarkably thermostable

Porcine trypsin was glycated with glucose and covalently immobilized through its carboxyl groups onto aminated glass beads to produce porcine immobilized glycated-trypsin (IGT). On incubation at 60 °C and pH 8, IGT retained its full activity for 8 h and 50% of its activity after 24 h. In comparison, under the same conditions porcine native trypsin lost 80% of its activity in 2 h and was completely inactivated in less than 4 h. The rate of autolysis of porcine glycated-trypsin at 37 °C was 40% that of native trypsin and with IGT there was no significant autolysis, even at elevated temperatures as high as 60 °C. Glycation significantly increased the stability of trypsin and immobilization also significantly increased the stability of trypsin. The remarkable thermostability of IGT is attributed to a synergistic effect when these two modifications are combined. Tryptic fragmentation of denatured proteins with IGT can be performed at 60 °C for shorter digestion times and with smaller amounts of enzyme than normally employed to achieve complete digestion with soluble forms of trypsin. Prior denaturation of proteins for tryptic digestion is not required with IGT as in situ denaturation and digestion can be achieved simultaneously at 60 °C with an enzyme:protein mass ratio as low as 1:1000.     

Heat stabilization produced by protein-protein association. A differential scanning calorimetric study of the heat denaturation of the trypsin-soybean trypsin inhibitor and trypsin-ovomucoid complexes.

The irreversible thermal denaturation of the association complexes of bovine beta-trypsin with soybean trypsin inhibitor or ovomucoid was observed with a differential scanning calorimeter. Association of trypsin with either inhibitor results in increased heat stability. The largest effect is observed with beta-trypsin and soybean trypsin inhibitor. At pH 6.7, first order rate constants (s-1) for denaturation at 72 degrees, determined at a heating rate of 10 degrees per min, are: beta-trypsin, 30 times 10-3; soybean trypsin inhibitor, 9 times 10-3; trypsin-soybean trypsin inhibitor complex, 0.4 times 10-3. Under equivalent conditions, rate constants for ovomucoid and trypsin-ovomucoid complex are 4 times 10-3 and 1 times 10-3 s-1, respectively. These changes in rate correspond to heat stabilization of trypsin equivalent to an increase of 16 and 9 degrees, respectively, in its observed denaturation temperature. Rate constants determined for beta-trypsin and trypsin-soybean trypsin inhibitor complex are independent of heating rate; those for soybean trypsin inhibitor and ovomucoid are a function of heating rate. This suggests that predenaturational conformational alterations may be important steps in the denaturation of the inhibitors. Activation energies for denaturation of the complexes and their components are all similar, averaging 70 kcal per mol. The large activation energies observed suggest that denaturation of the complexes is not rate-limited by their dissociation.     

Fluorescence and FTIR study of the pressure-induced denaturation of bovine pancreas trypsin.

The pressure denaturation of trypsin from bovine pancreas was investigated by fluorescence spectroscopy in the pressure range 0. 1-700 MPa and by FTIR spectroscopy up to 1000 MPa. The tryptophan fluorescence measurements indicated that at pH 3.0 and 0 degrees C the pressure denaturation of trypsin is reversible but with a large hysteresis in the renaturation profile. The standard volume changes upon denaturation and renaturation are -78 mL.mol-1 and +73 mL.mol-1, respectively. However, the free energy calculated from the data in the compression and decompression directions are quite different in absolute values with + 36.6 kJ.mol-1 for the denaturation and -5 kJ. mol-1 for the renaturation. For the pressure denaturation at pH 7.3 the tryptophan fluorescence measurement and enzymatic activity assays indicated that the pressure denaturation of trypsin is irreversible. Interestingly, the study on 8-anilinonaphthalene-1-sulfonate (ANS) binding to trypsin under pressure leads to the opposite conclusion that the denaturation is reversible. FTIR spectroscopy was used to follow the changes in secondary structure. The pressure stability data found by fluorescence measurements are confirmed but the denaturation was irreversible at low and high pH in the FTIR investigation. These findings confirm that the trypsin molecule has two domains: one is related to the enzyme active site and the tryptophan residues; the other is related to the ANS binding. This is in agreement with the study on urea unfolding of trypsin and the knowledge of the molecular structure of trypsin.     

胰蛋白酶与ANS的相互作用

利用荧光光谱法研究了在不同ｐＨ、压力及不同浓度的脲作用时荧光探针１,８－ＡＮＳ（１－ａｎｉｌｉｏｎｎａｐｈｔｈａｌｅｎｅ－８－ｓｕｌｆｏｎｉｃａｃｉｄ）与胰蛋白酶的相互作用．发现在低ｐＨ时ＡＮＳ可以结合到胰蛋白酶上,其中以ｐＨ２．０、３．０时结合最强．进一步的研究发现脲变性对胰蛋白酶结合ＡＮＳ的能力有很大的影响：１．５ｍｏｌ／Ｌ的脲即可使得胰蛋白酶结合ＡＮＳ的能力大大降低,但有趣的是即使高达４ｍｏｌ／Ｌ的脲对胰蛋白酶色氨酸残基荧光也无明显影响．另外,在ｐＨ猝变、脲变性、及逐渐改变压力时,胰蛋白酶色氨酸残基荧光和结合到胰蛋白酶分子上的ＡＮＳ的荧光的变化大不相同．上述结果暗示胰蛋白酶的色氨酸残基所在的区域和其结合ＡＮＳ的区域是两个不相同的区域．     

Production of egg yolk lysolecithin with immobilized phospholipase A2

Production of egg yolk lysolecithin was compared using free phospholipase A₂ (PLA₂) and immobilized PLA₂ in alginate-silicate sol-gel matrix. Choice of solvent, water content, calcium, and temperatures changed the activity of the free and immobilized PLA₂ a lot, owing to their effects on the catalytic properties of the enzyme as well as the conformational change of lecithin in ethanol-buffer mixture. Free PLA₂ shows typical microemulsion kinetics in ethanol-buffer system. The effect of the water content on the enzyme reaction was greatly influenced by the presence of calcium ion. In the absence of calcium ion, certain optimal water content for the production of lysolecithin always exists in the free PLA₂ reaction. However, with calcium ion, three distinctive regions were observed with free PLA₂ reactions. Initially, in the micro-aqueous region of the ethanol-buffer system with calcium ion, the hydrolysis activity of PLA₂ was proportional to the water content. Beyond the region, concave type of activity profiles were observed as the water content increases. As the water content increases further, the hydrolysis rate of the PLA₂ abruptly decreased by the phase separation. On the contrary, in case of immobilized enzyme, optimal water content for the production of lysolecithin exists regardless of the presence of calcium ion. The calcium ion was essential for achieving the maximum activity of both free and immobilized PLA₂. The addition of calcium ion not only affected the catalytic activity of the enzyme but also was necessary to improve the enzyme stability. As the immobilization of the enzyme remarkably increased thermal stability of the free enzyme, the immobilized PLA₂ is more desirable to be used in the production of various lysophospholipids. It was successfully reused over 250 h.     

Denaturation ofBacillus stearothermophilus α-amylase by urea and detergents

Denaturation ofBacillus stearothermophilus -amylase by urea and detergents was investigated for the purpose of understanding the mechanism of denaturation of this enzyme. The enzyme was extremely resistant to denaturation by detergents at 60° C, either in the presence or absence of added calcium. Addition of EDTA was necessary to obtain denaturation by detergents. The rate of denaturation of the -amylase by urea was strongly dependent on the incubation pH and presence or absence of calcium ions. Calcium-binding groups were shown to have pKa values of 5.5 for exogenous calcium and 4.7 to 4.8 for endogenous calcium. A mechanism is proposed for the denaturation ofBacillus stearothermophilus -amylase.     

Effect of Zinc Ions on Conformational Stability of Yeast Alcohol Dehydrogenase

Yeast alcohol dehydrogenase preparations were prepared with the conformational zinc ion removed (Apo-I YADH) and with both the conformational and catalytic zinc ions removed (Apo-II YADH). The unfolding of Apo-I YADH and Apo-II YADH during denaturation in urea solutions was then followed by fluorescence emission, circular dichroism, and second-derivative optical spectroscopies. Compared with the native enzyme, Apo-I YADH incurred some slight unfolding, and its stability against urea was markedly decreased, while Apo-II YADH incurred marked unfolding but contained residual ordered structure even at high urea concentrations. The results show that native YADH is more conformationally stable against urea denaturation than Apo-I YADH, indicating that the conformational Zn²⁺ plays an important role in stabilizing the conformation of the YADH molecule. However, unfolding of the region around the conformational zinc ion is shown not to be the rate limited step in the unfolding of the molecule by the fact that the unfolding and inactivation rate constants of native and Apo-I YADH are the same. It is suggested that the catalytic zinc ion is more important in maintaining the structure of YADH. YADH lost its cooperative unfolding ability after the zinc ions were removed. The shape of the transition curves of Apo-I YADH suggests the existence of an unfolding intermediate. For both native and Apo-I YADH, inactivation occurs at much lower urea concentrations than that needed to produce significant conformational changes of the enzyme molecule. At urea concentration above 4 M, the inactivation rate constants are much higher than those of the fast phase of the reaction of unfolding. These results support the suggestion of flexibility at the active site of the enzyme (C. L. Tsou (1986) Trends Biochem. Sci., 11, 427-429; (1993) Science, 262, 308-381).     

Calorimetric study of the thermal denaturation of beta-lactoglobulin in the presence of urea and phosphate ions]

The denaturation of beta-lactoglobulin in solution with different content of urea and phosphates has been studied calorimetrically. It has been shown that the increase of phosphate ion concentration in solution leads to an increase of beta-lactoglobulin stability, while increase of urea concentration leads to an opposite effect. The variation of these components in solution practically does not influence the value of the heat capacity increment of beta-lactoglobulin in the considered temperature region. Accordingly the denaturation enthalpy is a linear function of temperature whose slope does not differ for solution with urea concentration less than 4.4 M. However, the absolute value of denaturation enthalpy in these solutions at corresponding temperatures differs significantly due to the heat effect of additional urea solvation during transition to the denatured state. The latter leads to a decrease of the overall denaturation enthalpy and, as a result, a shift of the enthalpy plot to higher temperatures providing conditions for studying the thermodynamic and structural characteristics of the molecule in the cold denatured-state.     

Fast proteolytic digestion coupled with organelle enrichment for proteomic analysis of rat liver

The use of an acid-labile surfactant as an alternative to urea denaturation allows for same-day proteolytic digestion and fast cleanup of cellular lysate samples. Homogenized rat liver tissue was separated into four fractions enriched in nuclei, mitochondria, microsomes (remaining organelles), and cytosol. Each subcellular fraction was then subjected to proteolytic digestion with trypsin for 2 h after denaturing with an acid-labile surfactant (ALS), separated by nanoflow reversed phase HPLC, and mass analyzed by tandem mass spectrometry in a 3-D ion trap. The results obtained from ALS denaturation for both organelle enrichment and whole cell lysate samples were comparable to those obtained from aliquots of the same samples treated by reduction, alkylation, and urea denaturation. Each method resulted in a similar number of peptides (694 for urea, 674 for ALS) and proteins (225 for urea, 229 for ALS) identified, with generally the same proteins (47% overlap) identified. As expected, organelle enrichment enabled the identification of more proteins (66% more with urea, 60% more with ALS) compared to a whole cell lysate. With organelle enrichment, the number of proteins with equal or increased sequence coverage went up by 73% with urea and 67% with ALS compared to the whole cell lysate. Additional information regarding the subcellular location of many proteins is obtained by organelle enrichment. While organelle enrichment is demonstrated with a bottom-up proteomics approach, it should be easily amenable to top-down proteomics approaches.     

Thermodynamics of denaturation of hisactophilin, a beta-trefoil protein

Hisactophilin is a histidine-rich pH-dependent actin-binding protein from Dictyostelium discoideum. The structure of hisactophilin is typical of the beta-trefoil fold, a common structure adopted by diverse proteins with unrelated primary sequences and functions. The thermodynamics of denaturation of hisactophilin have been measured using fluorescence- and CD-monitored equilibrium urea denaturation curves, pH-denaturation, and thermal denaturation curves, as well as differential scanning calorimetry. Urea denaturation is reversible from pH 5.7 to pH 9.7; however, thermal denaturation is highly reversible only below pH approximately 6.2. Reversible denaturation by urea and heat is well fit using a two-state transition between the native and the denatured states. Urea denaturation curves are best fit using a quadratic dependence of the Gibbs free energy of unfolding upon urea concentration. Hisactophilin has moderate, roughly constant stability from pH 7.7 to pH 9.7; however, below pH 7.7, stability decreases markedly, most likely due to protonation of histidine residues. Enthalpic effects of histidine ionization upon unfolding also appear to be involved in the occurrence of cold unfolding of hisactophilin under relatively mild solution conditions. The stability data for hisactophilin are compared with data on hisactophilin function, and with data for two other beta-trefoil proteins, human interleukin-1beta, and basic fibroblast growth factor.