Binding Of Penicillin Research Articles

Mutagenesis and structural analysis reveal the CTX-M β-lactamase active site is optimized for cephalosporin catalysis and drug resistance

CTX-M β-lactamases are a widespread source of resistance to β-lactam antibiotics in Gram-negative bacteria. These enzymes readily hydrolyze penicillins and cephalosporins, including oxyimino-cephalosporins such as cefotaxime. To investigate the preference of CTX-M enzymes for cephalosporins, we examined eleven active-site residues in the CTX-M-14 β-lactamase model system by alanine mutagenesis to assess the contribution of the residues to catalysis and specificity for the hydrolysis of the penicillin, ampicillin, and the cephalosporins cephalothin and cefotaxime. Key active site residues for class A β-lactamases, including Lys73, Ser130, Asn132, Lys234, Thr216, and Thr235, contribute significantly to substrate binding and catalysis of penicillin and cephalosporin substrates in that alanine substitutions decrease both kcat and kcat/KM. A second group of residues, including Asn104, Tyr105, Asn106, Thr215, and Thr216, contribute only to substrate binding, with the substitutions decreasing only kcat/KM. Importantly, calculating the average effect of a substitution across the 11 active-site residues shows that the most significant impact is on cefotaxime hydrolysis while ampicillin hydrolysis is least affected, suggesting the active site is highly optimized for cefotaxime catalysis. Furthermore, we determined X-ray crystal structures for the apo-enzymes of the mutants N106A, S130A, N132A, N170A, T215A, and T235A. Surprisingly, in the structures of some mutants, particularly N106A and T235A, the changes in structure propagate from the site of substitution to other regions of the active site, suggesting that the impact of substitutions is due to more widespread changes in structure and illustrating the interconnected nature of the active site.

Impacts of NaHCO3 on β-Lactam Binding to PBP2a Protein Variants Associated with the NaHCO3-Responsive versus NaHCO3-Non-Responsive Phenotypes.

Methicillin-resistant Staphylococcus aureus (MRSA) regulates resistance to β-lactams via preferential production of an alternative penicillin-binding protein (PBP), PBP2a. PBP2a binds many β-lactam antibiotics with less affinity than PBPs which are predominant in methicillin-susceptible (MSSA) strains. A novel, rather frequent in vitro phenotype was recently identified among clinical MRSA bloodstream isolates, termed “NaHCO3-responsiveness”. This phenotype features β-lactam susceptibility of certain MRSA strains only in the presence of NaHCO3. Two distinct PBP2a variants, 246G and 246E, have been linked to the NaHCO3-responsive and NaHCO3-non-responsive MRSA phenotypes, respectively. To determine the mechanistic impact of PBP2a variants on β-lactam susceptibility, binding profiles of a fluorescent penicillin probe (Bocillin-FL) to each purified PBP2a variant were assessed and compared to whole-cell binding profiles characterized by flow cytometry in the presence vs. absence of NaHCO3. These investigations revealed that NaHCO3 differentially influenced the binding of the fluorescent penicillin, Bocillin-FL, to the PBP2a variants, with binding intensity and rate of binding significantly enhanced in the 246G compared to the 246E variant. Of note, the NaHCO3-β-lactam (oxacillin)-responsive JE2 strain, which natively harbors the 246G variant, had enhanced Bocillin-FL whole-cell binding following exposure to NaHCO3. This NaHCO3-mediated increase in whole-cell Bocillin-FL binding was not observed in the NaHCO3-non-responsive parental strain, COL, which contains the 246E PBP2a variant. Surprisingly, genetic swaps of the mecA coding sites between JE2 and COL did not alter the NaHCO3-enhanced binding seen in JE2 vs. COL. These data suggest that the non-coding regions of mecA may be involved in NaHCO3-responsiveness. This investigation also provides strong evidence that the NaHCO3-responsive phenotype in MRSA may involve NaHCO3-mediated increases in both initial cell surface β-lactam binding, as well as ultimate PBP2a binding of β-lactams.

Combining Electrochemiluminescence Detection with Aptamer-Gated Indicator Releasing Mesoporous Nanoparticles Enables ppt Sensitivity for Strip-Based Rapid Tests.

The combination of electrogenerated chemiluminescence (ECL) and aptamer‐gated indicator delivering (gAID) magnetic mesoporous silica nanoparticles embedded into glass fibre paper functionalised with poly(ethyleneglycol) and N‐(3‐triethoxysilylpropyl)diethanolamine allowed the development of a rapid test that detects penicillin directly in diluted milk down to 50±9 ppt in <5 min. Covalent attachment of the aptamer “cap” to the silica scaffold enabled pore closure through non‐covalent electrostatic interactions with surface amino groups, while binding of penicillin led to a folding‐up of the aptamer thus releasing the ECL reporter Ru(bpy)3 2+ previously loaded into the material and letting it be detected after lateral flow by a smartphone camera upon electrochemical excitation with a screen printed electrode inserted into a 3D‐printed holder. The approach is simple, generic and presents advantages with respect to sensitivity, measurement uncertainty and robustness compared with conventional fluorescence or electrochemical detection, especially for point‐of‐need analyses of challenging matrices and analytes at ultra‐trace levels.

Re-construction of Co-expression Network of Genes Involved in Bacterial Cell Wall Synthesis and Their Role in Penicillin Resistance

Background: Peptidoglycan (Murein), which consists of disaccharide and amino acid chain subunits, has a key role in bacterial survival and ranks first in the line defense system against drug therapy. In addition, the transpeptidase enzyme plays an important role in cross-linking in bacterial cell walls. In Escherichia coli bacteria, cross-linking happens by proteins that have a D-D transpeptidase role and bond two amino acids of D-alanine together. These proteins are characterized by their affinity for and binding of penicillin thus they are called penicillin-binding proteins (PBPs). It should be noted that this bonding formation is prevented by the beta-lactam family as they have a similar structure to the above-mentioned proteins. The product of the idtD gene by characteristics such as L-D transpeptidase can catalyze the peptidoglycan structure in the bacterial cell wall in the presence of beta-lactam antibiotics. Methods: In this study, around 426 interactions were identified between genes and approximately 20 genes with a key role in the process of bacterial cell wall synthesis by the reconstruction of 44 genes involved in bacterial cell wall synthesis. Results: The idtD gene locus at the reconstructed network clearly shows that its catalytic activity is the side activity, and there won’t be a lag or disturbance in the procedure cell wall synthesis by removing it from the cycle. However, this side process causes the strengthening of the bacterial cell wall synthesis process against disorders arising by the presence of beta-lactam antibiotics. Conclusions: These five genes in E. coli that furnish L-D transpeptidase properties include IdtA, IdtC, IdtD, IdtE, and mrdA out of which, IdtD is the most important gene in this process.

Proliferation rate and penicillin binding of enterococcal isolates from patients with immunosupression.

Low proliferation rate of bacterial populations was recently assumed to be a reason for higher resistance to antibiotics and appearance of many chronic infections. Slowly growing populations, called 'small colony variants' (SCVs) have been described in many bacterial species to make from as low as 0·02% up to 46% of population. Thirty enterococcal strains from urine and faeces of renal transplant recipients with asymptomatic, insignificant bacteriuria were studied. Growth characteristics were estimated by microculture and OD reading after 1, 3 and 5h of culture. At the same time, penicillin binding and changes of aggregation of the cells were analysed by flow cytometry. The results of our study showed high diversity of the proliferation rates among studied isolates. Based on proliferation rates and aggregation, six of studied strains (20%) could be considered as SCVs-like. Significantly lower binding of penicillin was also observed for these SCV-like strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides relevant information about prevalence of enterococcal strains with low proliferation rate (likely small colony variant (SCV)) among kidney transplant recipients. Percentage of such strains in this cohort was relatively high (20%). Additionally, penicillin binding of these strains measured even at the beginning of proliferation (after 1 and 3h of incubation), was significantly lower than among other strains. Finally, all of them were determined as penicillin resistant, with minimal inhibitory concentration value above 256μgml-1 . As the risk of systemic infections caused by such strains is probably higher than in case of other strains, screening for the SCVs in this group of patients should be recommended.

Antibacterial Drug Releasing Materials by Post-Polymerization Surface Modification

Functional materials are available by the post-polymerization surface modification of diverse polymers in a three-step process mediated, firstly, by carbene insertion chemistry, secondly, by diazonium coupling, and thirdly by modification with a remotely tethered spiropyran unit, and these materials may be used for the reversible binding and release of Penicillin V. Surface loading densities of up to 0.19[Formula: see text]mmol/g polymer are achievable, leading to materials with higher loading densities and release behavior relative to unmodified controls, and observable antibacterial biocidal activity.

Fabrication of a Single Layer and Bilayer Potentiometric Biosensors for Penicillin by Galvanostatic Entrapment of Penicillinase into Polypyrrole Films

AbstractA single layer and bilayer potentiometric biosensors for the detection of penicillin have been developed. The favourable conditions that were established for the polypyrrole‐penicillinase ((PPy‐P’nase) single layer biosensor were 0.03 M pyrrole, 50 U/mL P’nase, 0.01 M penicillin, applied current density of 0.9 mA/cm2 and a polymerisation time of 40 s. The optimum conditions for the formation of the outer layer of the bilayer were: (a) 0.1 M Py, 19 U/mL P’nase, 0.01 M pen, current density of 0.9 mA/cm2 and a polymerisation time of 40 s. The minimum detectable penicillin concentration with the bilayer potentiometric biosensor was 0.3 µM and the linear concentration range was 7.5–146 µM. The average percentage recovery of penicillin that was found in amoxycillin 500 mg was 113±24 %. The determination of penicillin in milk was fraught with problems of non‐specific binding of penicillin to the milk.

Sensitization of Staphylococcus aureus to Methicillin and Other Antibiotics In Vitro and In Vivo in the Presence of HAMLET

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a protein-lipid complex from human milk with both tumoricidal and bactericidal activities. HAMLET exerts a rather specific bactericidal activity against some respiratory pathogens, with highest activity against Streptococcus pneumoniae, but lacks activity against most other bacterial pathogens, including Staphylococci. Still, ion transport associated with death in S. pneumoniae is also detected to a lower degree in insensitive organisms. In this study we demonstrate that HAMLET acts as an antimicrobial adjuvant that can increase the activity of a broad spectrum of antibiotics (methicillin, vancomycin, gentamicin and erythromycin) against multi-drug resistant Staphylococcus aureus, to a degree where they become sensitive to those same antibiotics, both in antimicrobial assays against planktonic and biofilm bacteria and in an in vivo model of nasopharyngeal colonization. We show that HAMLET exerts these effects specifically by dissipating the proton gradient and inducing a sodium-dependent calcium influx that partially depolarizes the plasma membrane, the same mechanism induced during pneumococcal death. These effects results in an increased cell associated binding and/or uptake of penicillin, gentamicin and vancomycin, especially in resistant stains. Finally, HAMLET inhibits the increased resistance of methicillin seen under antibiotic pressure and the bacteria do not become resistant to the adjuvant, which is a major advantageous feature of the molecule. These results highlight HAMLET as a novel antimicrobial adjuvant with the potential to increase the clinical usefulness of antibiotics against drug resistant strains of S. aureus.

Influence of salt ions on binding to molecularly imprinted polymers

Salt ions were found to have an influence on template binding to two model molecularly imprinted polymers (MIPs), targeted to penicillin G and propranolol, respectively, in water-acetonitrile mixtures. Water was detrimental to rebinding of penicillin G whereas propranolol bound in the entire water-acetonitrile range tested. In 100% aqueous solution, 3-M salt solutions augmented the binding of both templates. The effects followed the Hofmeister series with kosmotropic ions promoting the largest increase. Binding was mainly of a non-specific nature under these conditions. In acetonitrile containing low amounts of water, the specific binding to the MIPs increased with the addition of salts. Binding of penicillin G followed the Hofmeister series while an ion-exchange mechanism was observed for propranolol. The results suggest that hydration of kosmotropic ions reduces the water activity in water-poor media providing a stabilizing effect on water-sensitive MIP-template interactions. The effects were utilized to develop a procedure for molecularly imprinted solid-phase extraction (MISPE) of penicillin G from milk with a recovery of 87%.

A tryptophan residue is identified in the substrate binding of penicillin G acylase from Kluyvera citrophila

Penicillin acylases are important enzymes in pharmaceutical industry for the production of semi-synthetic β-lactam antibiotics via the key intermediate 6-aminopenicillanic acid. The penicillin G acylase purified from Kluyvera citrophila ( KcPGA) on modification with tryptophan-specific reagents such as N-bromo succinamide (NBS) and 2-hydroxy 5-nitrobenzylbromide (HNBB) showed partial loss of activity and substrate protection. Various solute quenchers and substrate were used to probe the microenvironment of the putative reactive tryptophan through fluorescence quenching. Homology modeling of KcPGA structure has been carried out. Docking substrate on this modeled KcPGA structure identifies the tryptophan residue that is directly influenced by substrate binding. To confirm the biological significance of this particular tryptophan, we did a sequence comparison of PGAs from various organisms. The sequence alignment clustered the matches into two sets, those closer to (>40% identical) KcPGA and had the tryptophan of interest present in them formed the first set, while those less identical (<30%) to KcPGA and the particular tryptophan absent in them formed the second set. It is clear from the reported kinetic parameters of representative members of these two sets that the affinity for penicillin G (penG) of the former class is several times better. Thus, based on our studies we suggest that the tryptophan residue in the identified position is important for binding substrate penG by the acylases.

Exploring the molecular basis of the enantioselective binding of penicillin G acylase towards a series of 2-aryloxyalkanoic acids: A docking and molecular dynamics study

In the present paper, molecular modeling studies were undertaken in order to shed light on the molecular basis of the observed enantioselectivity of penicillin G acylase (PGA), a well known enzyme for its industrial applications, towards 16 racemic 2-aryloxyalkanoic acids, which have been reported to affect several biological systems. With this intention docking calculations and MD simulations were performed. Docking results indicated that the ( S)-enantiomers establish several electrostatic interactions with SerB1, SerB386 and ArgB263 of PGA. Conversely, the absence of specific polar interactions between the ( R)-enantiomers and ArgB263 seems to be the main reason for the different binding affinities observed between the two enantiomers. Results of molecular dynamics simulations demonstrated that polar interactions are responsible for both the ligand affinity and PGA enantiospecificity. Modeling calculations provided possible explanations for the observed enantioselectivity of the enzyme that rationalize available experimental data and could be the basis for future protein engineering efforts.

Chemical modifications of the active site of Streptomyces R61 DD-carboxypeptidase.

The DD-carboxypeptidase of Streptomyces R61 is an exocellular enzyme related to the bacterial peptidoglycan cross-linking enzymes, and, like them, is inhibited by penicillin. The active-site reagents methanesulfonyl fluoride and diisopropylfluorophosphate inhibit catalytic activity and binding of penicillin G indicating the involvement of a serine residue in both processes. For methanesulfonyl fluoride the second-order rate constant (0.7 M-1 min-1) is comparable to that of classical serine proteases. For diisopropylfluorophosphate, which binds to the enzyme stoichiometrically, the second-order rate constant (1.5 M-1 min-1) is at least two orders of magnitude smaller. The arginine-specific reagents methylglyoxal, 2,3-butanedione and phenylglyoxal inactive DD-carboxypeptidase in borate buffer with second-order rate constants of 70, 70 and 120 M-1 min-1, respectively. Inactivation correlates with stoichiometric binding to the enzyme. Peptidase and esterase activities are similarly affected, suggesting that substrate binding in both cases requires an arginine-carboxyl group interaction. Penicillin binding is also inhibited, but the degree of inhibition depends on the alpha-dicarbonyl side chain. Binding of alpha-dicarbonyls to DD-carboxypeptidase facilitates subsequent binding of diisopropylfluorophosphate suggesting that interaction of these compounds with the active site might induce a conformational change on the enzyme making the serine residue more accessible to the modifying reagent.

Interactions of Two Amphiphilic Penicillins with Myoglobin in Aqueous Buffered Solutions: A Thermodynamic and Spectroscopy Study

The interactions and complexation process of the amphiphilic penicillins sodium cloxacillin and sodium dicloxacillin with horse myoglobin in aqueous buffered solutions of pH 4.5 and 7.4 have been examined by equilibrium dialysis, zeta-potential, isothermal titration calorimetry (ITC) and UV-Vis absorbance techniques. A more opened structure of the protein molecules is detected as a consequence of the reduction of pH from 7.4 to 4.5. Binding isotherms and derived Hill coefficients reflect a cooperative binding behavior. Gibbs energies of binding per mole of drug were obtained from equilibrium dialysis data and compared with those derived from the zeta potential taking into account cooperativity. DeltaGads degrees values so obtained are large and negative at low concentrations where binding to the "high-energy" sites occurs and decreases with the drug concentration. The enthalpies of binding have been obtained from ITC and are small and exothermic so that the Gibbs energies of binding are dominated by large increases in entropy consistent with hydrophobic interactions. Other thermodynamic quantities of the binding mechanism, that is, entropy, DeltaSITCi, Gibbs energy, DeltaGITCi, the binding constant, KITCi, and the number of binding sites, ni, were also obtained, confirming the above results. From ITC data and following a theoretical model, the number of bound and free penicillin molecules was calculated, being higher at pH 4.5 than at pH 7.4. The binding of penicillin causes a conformational transition on protein structure as a consequence of the resulting intramolecular repulsion between the penicillin molecules bound to the protein. Thermodynamic quantites (the Gibbs energy of the transition in water, DeltaGw degrees , and in a hydrophobic environment, DeltaGhc degrees) of the denaturation process were calculated, indicating that at pH 4.5 some of the histidine residues are protonated, becoming accessible to solvent and giving rise to a more opened protein structure.

Binding properties of human albumin modified by covalent binding of penicillin

Derivatisation of lysine residues in human albumin was performed in vitro by reaction with penicillin G. This modification reaction has been reported to occur in patients treated with high dosages of the antibiotic. The structure of the modified protein was characterised by mass spectrometry and circular dichroism. The number of the lysine residues involved depends on the time of incubation and on the drug/protein molar ratio. The secondary structure of the modified protein does not change significantly with respect to the native protein. Furthermore, the binding properties of the modified albumin were characterised by CD spectroscopy. Phenylbutazone, diazepam and bilirubin, known to bind to specific binding areas, were used as markers. A decrease of the affinity to the high-affinity binding sites was observed after the modification.

Mutational analysis of metallo-beta-lactamase CcrA from Bacteroides fragilis.

In an effort to evaluate the roles of Lys184, Asn193, and Asp103 in the binding and catalysis of metallo-beta-lactamase CcrA from Bacteroides fragilis, site-directed mutants of CcrA were generated and characterized using metal analyses, CD spectroscopy, and kinetic studies. Three Lys184 mutants were generated where the lysine was replaced with alanine, leucine, and glutamate, and the analysis of these mutants indicates that Lys184 is not greatly involved in binding of cephalosporins to CcrA; however, this residue does have a significant role in binding of penicillin G. Three Asn193 mutants were generated where the asparagine was replaced with alanine, leucine, and aspartate, and these mutants exhibited <4-fold decrease in k(cat), suggesting that Asn193 does not play a large role in catalysis. However, stopped-flow visible kinetic studies showed that the Asn193 mutants exhibit a slower substrate decay rate and no change in the product formation rate as compared with wild-type CcrA. These results support the proposed role of Asn193 in interacting with and activating substrate during catalysis. Two Asp103 mutants were generated where the aspartate was replaced with serine and cysteine. The D103C and D103S mutants bind the same amount of Zn(II) as wild-type CcrA and exhibited a 10(2)-fold and 10(5)-fold decrease in activity, respectively. Results from solvent isotope, proton inventory, and rapid-scanning visible studies suggest that Asp103 plays a role in generating the enzyme intermediate but does not donate a proton to the enzyme intermediate during the rate-limiting step of the catalytic mechanism.

Beaded reactive polymers. 3. Effect of triacrylates as crosslinkers on the physical properties of glycidyl methacrylate copolymers and immobilization of penicillin G acylase.

Various glycidyl methacrylate (GMA) copolymers were synthesized by suspension polymerization, using pentaerythritol triacrylate (PETA), trimethylolpropane triacrylate (TMPTA), and trimethylolpropane trimethacrylate (TRIM) as crosslinking comonomers. These copolymers were evaluated for the immobilization of penicillin G acylase. Broad pore-size distribution that was observed was in the range 5-300 nm. Both surface area and pore volume increased with increase in the mole fraction of crosslinking comonomer (increasing crosslink density). The pore volume of the copolymers was more than doubled by including lauryl alcohol as porogen. Binding of penicillin G acylase (PGA) was quantitative on highly crosslinked copolymers. The expression of bound PGA was better on the relatively more hydrophilic GMA-TMPTA and GMA-PETA copolymer supports compared to the GMA-TRIM copolymers. Among the different copolymers studied, GMA-TMPTA copolymer 7411 exhibited highest activity of immobilized penicillin G acylase (167.4 IU/g) with 35.1% expression.

Penicillin-binding protein sensitive to cephalexin in sporulation of Bacillus cereus

Cephalexin, cefaclor, cefadroxil, and cefotaxime strongly inhibited sporulation of Bacillus cereus ts-4 at 1 μg/ml. Cephalexin was most inhibitory on the sporulation of B. cereus when the antibiotic was added at 3 h after induction of sporulation by nutrient downshift technique. Examination of 4′,6-diamidino-2-phenylindole-stained cells by fluorescence-phase contrast microscopy showed that cephalexin inhibited the formation of asymmetric septum. By using [ 3H]penicillin, eight penicillin-binding proteins (PBPs) were detected from the cells of B. cereus ts-4. Among them, four PBPs were also detected in sporulating cells. Affinity of cephalexin to PBPs were measured indirectly by competition for subsequent binding of radioactive penicillin G. Cephalexin strongly bound to PBP 4 with molecular weight of 72,000 in sporulating cells.

Persistent acylation of high-molecular-weight penicillin-binding proteins by penicillin induces the postantibiotic effect in Streptococcus pyogenes.

Penicillin at 10X MIC induced a postantibiotic effect (PAE) of 2.1 h in Streptococcus pyogenes. Progressive increases in the densities of penicillin-binding proteins (PBPs) 1-3 of the bacterium were detected at 30, 60, and 90 min during the postantibiotic phase. The increase in colony-forming units during this phase paralleled the kinetics of incorporation of lysine into proteins, suggesting that growth was triggered by de novo synthesis of PBPs. The question was raised as to whether the progressive increases in densities of PBPs were due to the restoration of preexisting PBPs or to synthesis of new PBPs. With 10X MIC of clindamycin to inhibit PBP synthesis during the postantibiotic phase, the temporal increase in densities of PBPs 1-3 were totally inhibited. These results suggest that the PAE of penicillin in S. pyogenes is caused by irreversible binding of penicillin to PBPs 1-3 and represents the time necessary for synthesis of new PBPs required for normal growth.

Site-directed mutagenesis of the mecA gene from a methicillin-resistant strain of Staphylococcus aureus

The mecA-27r gene from Staphylococcus aureus 27r encodes penicillin-binding protein 2a (PBP2a-27r), which causes this strain to be methicillin resistant. Removal or replacement of the N-terminal transmembrane domain had no effect on binding of penicillin, but removal of portions of the putative transglycosylase domain (144, 245, or 341 amino acids after the transmembrane region) destroyed penicillin-binding activity. The SXXK, SXN, and KSG motifs, present in all penicillin-interacting enzymes, were found in the expected linear spatial arrangement within the putative transpeptidase region of PBP2a-27r. Alterations of amino acids in all three of these motifs resulted in elimination of penicillin-binding activity, confirming their roles in the interaction with penicillin.

Facilitation of penicillin haptenation to serum proteins

Traditionally, penicillin binding to serum proteins was believed to be a passive chemical process; however, it appears to be facilitated by serum factors. The objectives of this in vitro investigation were to examine facilitated penicillin haptenation, to study the kinetics of haptenation, and to determine the nature of haptenation-facilitating factors. The model involved addition of [3H]benzylpenicillin to serum or albumin solutions (at pH 7.3 to 7.4) and incubation at 37 degrees C for up to 72 h. The extent of penicillin binding to proteins in serum was found to be four- to fivefold higher than with solutions having comparable concentrations of purified albumin, total protein, or total immunoglobulin. Ultrafiltration of serum reduced penicillin binding to serum proteins substantially. An ultrafiltrable haptenation-facilitating factor(s) was found to be less than 0.5 kDa but was not calcium or magnesium. Finally, the extent of penicillin binding was related to albumin purity, as binding substantially increased with albumin purity. These findings suggest that there is a factor(s) in serum that facilitates covalent binding of penicillin to serum proteins. The factor(s) can be removed and then restored to increase penicillin binding to albumin. It appears that at least one component of the facilitation factor is less than 0.5 kDa, which suggests that it is not a peptide and that it is some simple serum component other than calcium or magnesium.