Inhibition Of Calcium Phosphate Precipitation Research Articles

Structure, stability, and mechanism of dextran–CPP–Ca2+ conjugates: A novel high-efficiency calcium ion delivery system

Calcium has limited bioavailability because of the formation of calcium phosphate deposits in the gastrointestinal tract. In this study, we prepared a dextran-casein phosphopeptide (CPP)–Ca2+ delivery system and evaluated for Ca2+ binding mechanism, structure, stability, and sustained release of Ca2+ and assessed inhibition of calcium phosphate precipitation. The results revealed that Ca2+ binds to dextran–CPP through the phosphate, carboxyl, and amino groups and forms crystal clusters. Furthermore, compared with single polymer CPP–Ca2+ conjugates, copolymer dextran–CPP–Ca2+ conjugates exhibited improved stability at various conditions (pH, temperature, and coexisting food), efficiently reduced the calcium phosphate precipitation, and improved sustained-release of Ca2+. Collectively, dextran–CPP–Ca2+ conjugates can be an efficient Ca2+ delivery system.

Development of a cyclic-inverso AHSG/Fetuin A-based peptide for inhibition of calcification in osteoarthritis

Ectopic calcification is an important contributor to chronic diseases, such as osteoarthritis. Currently, no effective therapies exist to counteract calcification. We developed peptides derived from the calcium binding domain of human Alpha-2-HS-Glycoprotein (AHSG/Fetuin A) to counteract calcification. A library of seven 30 amino acid (AA) long peptides, spanning the 118 AA Cystatin 1 domain of AHSG, were synthesized and evaluated in an in vitro calcium phosphate precipitation assay. The best performing peptide was modified (cyclic, retro-inverso and combinations thereof) and evaluated in cellular calcification models and the rat Medial Collateral Ligament Transection + Medial Meniscal Tear (MCLT+MMT) osteoarthritis model. A cyclic peptide spanning AA 1-30 of mature AHSG showed clear inhibition of calcium phosphate precipitation in the nM-pM range that far exceeded the biological activity of the linear peptide variant or bovine Fetuin. Biochemical and electron microscopy analyses of calcium phosphate particles revealed a similar, but distinct, mode of action in comparison with bFetuin. A cyclic-inverso variant of the AHSG 1-30 peptide inhibited calcification of human articular chondrocytes, vascular smooth muscle cells and during osteogenic differentiation of bone marrow derived stromal cells. Lastly, we evaluated the effect of intra-articular injection of the cyclic-inverso AHSG 1-30 peptide in a rat osteoarthritis model. A significant improvement was found in histopathological osteoarthritis score and animal mobility. Serum levels of IFNγ were found to be lower in AHSG 1-30 peptide treated animals. The cyclic-inverso AHSG 1-30 peptide directly inhibits the calcification process and holds the potential for future application in osteoarthritis.

Self-association of caseinomacropeptide in presence of CaCl2 at neutral pH: Calcium binding determination

The caseinomacropeptide (CMP) is a bioactive peptide produced during cheese making. It is found in abundance in whey. CMP aqueous solutions allow the incorporation of large amounts of CaCl2 but the mechanism of calcium-CMP interactions are unknown. In order to evaluate its calcium binding capacity, the following techniques were performed: Dynamic Light Scattering (DLS), Fourier Transform Infrared spectroscopy (FTIR), dialysis, conductivity, precipitation of CaCl2/CMP complex by ethanol, electrochemical Ca2+ binding isotherms, and inhibition of calcium phosphate precipitation. One mole of CMP can bind 9 mol of calcium, and the CMP self-assembles as a hexameric form. A model is proposed to explain the CMP self-association in presence of CaCl2.

Dispersion of hydroxyapatite nanocrystals stabilized by polymeric molecules bearing carboxy and sulfo groups

Calcium phosphate buildup is important for biological processes (e.g., bones and teeth growth) and negatively affects the efficiency and functioning of water systems (e.g., cooling water systems and boilers) as a result of scale buildup by salts precipitation. Thus, stable aqueous dispersions of calcium phosphate particles can effectively prevent the formation and buildup of scale in water systems. In this study, the stabilization of calcium phosphate nanocrystals (NCs) was studied by forming a supersaturated solution in the presence of poly acrylic acid/2-acrylamide-2-methylpropane sulfonic acid p(AA/AMPS) containing carboxy and sulfo group functionalities. Calcium phosphate NCs were formed and subsequently aggregated in a supersaturated aqueous solution containing 2-phosphonobutane-1,2,4-tricarboxylic acid without any copolymers. Aggregation of calcium phosphate NCs was suppressed in the presence of polymeric molecules containing carboxy groups, whereas molecules bearing sulfo groups were ineffective in dispersing calcium phosphate NCs. However, copolymers bearing both functional groups enhanced the dispersibility of calcium phosphate NCs. Using a combination of light scattering and surface charge measurements along with electron imaging, we found that the inhibition of calcium phosphate precipitation originates from hydroxyapatite nanocrystals (HANCs) with a size smaller than 100 nm in solution. While the carboxy groups in the copolymer adsorbed on the surface of the HANCs, the sulfo groups provided these species with an overall negative surface charge, thereby increasing their colloidal stability via electrostatic repulsion. These results indicated that the aggregation of the HANCs can be effectively hindered using sulfo/carboxy bifunctional copolymers.

Impact of iron oxide (rust) on the performance of calcium phosphate scale inhibitors for industrial water systems

The inhibition of calcium phosphate precipitation by homo- and copolymers containing different functional groups has been examined in aqueous solution. It has been found that polymer performance as calcium phosphate inhibitor depends upon polymer architecture. The influence of iron oxide (Fe2O3, rust) on the performance of calcium phosphate inhibitors has also been investigated. Results show that addition of small amount (10 mg to 100 mg) of Fe2O3 to the calcium phosphate supersaturated solution exhibits marked antagonistic effect on inhibitor performance. Various mechanism(s) have been proposed to explain the negative impact of Fe2O3 on inhibitor performance.

Toward a Structure Determination Method for Biomineral-Associated Protein Using Combined Solid- State NMR and Computational Structure Prediction

Protein-biomineral interactions are paramount to materials production in biology, including the mineral phase of hard tissue. Unfortunately, the structure of biomineral-associated proteins cannot be determined by X-ray crystallography or solution nuclear magnetic resonance (NMR). Here we report a method for determining the structure of biomineral-associated proteins. The method combines solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. In addition, the algorithm is able to identify lattice geometries most compatible with ssNMR constraints, representing a quantitative, novel method for investigating crystal-face binding specificity. We use this method to determine most of the structure of human salivary statherin interacting with the mineral phase of tooth enamel. Computation and experiment converge on an ensemble of related structures and identify preferential binding at three crystal surfaces. The work represents a significant advance toward determining structure of biomineral-adsorbed protein using experimentally biased structure prediction. This method is generally applicable to proteins that can be chemically synthesized.

Hierarchical Role of Fetuin-A and Acidic Serum Proteins in the Formation and Stabilization of Calcium Phosphate Particles

The serum protein fetuin-A is a potent systemic inhibitor of soft tissue calcification. Fetuin-A is highly effective in the formation and stabilization of protein-mineral colloids, referred to as calciprotein particles (CPPs). These particles ripen in vitro in a two-step process, indicated by a morphological conversion from spheres to larger prolate ellipsoids. Using a combined light scattering and electron microscopic imaging approach we determined that the second-stage particles resulted from a highly anisotropic outgrowth of the first-stage particles. Electron microscopy of ascites fluid from a patient with calcifying peritonitis revealed particles reminiscent of secondary CPPs. Thus, CPPs form in the body and undergo the two-step ripening at least in pathological conditions. Unlike in vitro generated CPPs, ascites-derived CPPs contained little fetuin-A but large amounts of albumin. This prompted us to study the role of fetuin-A combined with other serum proteins in CPP formation. Fetuin-A was indispensable for primary CPP formation. Albumin and acidic proteins in general greatly enhanced the fetuin-A triggered formation of secondary CPPs and, thus, substituted substantial amounts of fetuin-A without loss of inhibition of calcium phosphate precipitation. Thus, direct mineral deposition from solute in the body is unlikely even at low fetuin-A serum levels as long as sufficient bulk acidic protein is available. Collectively fetuin-A and other acidic bulk plasma proteins may be considered as mineral chaperones mediating the stabilization, safe transport, and clearance in the body of calcium and phosphate as colloidal complexes, thus, preventing ectopic calcification.

The fallacy of the calcium-phosphorus product

Scattered through the practice of medicine are dogmas with little or no scientific basis. One of these is the product of the serum calcium and phosphorus concentrations, the so-called calcium-phosphorus product or Ca x P. The assumption that ectopic calcification will occur when the product of the serum calcium and phosphorus concentrations exceeds a particular threshold has become standard practice in nephrology even though there is little scientific basis. Experimental support is lacking, the chemistry underlying the use of the product is oversimplified and the concept that ectopic calcification is simply the result of supersaturation is biologically flawed. The evidence that the Ca x P is an independent risk factor for mortality and morbidity is also questionable. Although ectopic calcification can occur in many sites, this review will focus on vascular calcification, as it is the most common site and the site most likely to affect patient outcomes.

Inhibition of calcium phosphate precipitation under environmentally-relevant conditions

Precipitation of Ca phosphates plays an important role in controlling P activity and availability in environmental systems. The purpose of this study was to determine inhibitory effects on Ca phosphate precipitation by Mg(2+), SO(4)(2-), CO(3)(2-), humic acid, oxalic acid, biogenic Si, and Si-rich soil clay commonly found in soils, sediments, and waste streams. Precipitation rates were determined by measuring decrease of P concentration in solutions during the first 60 min; and precipitated solid phases identified using X-ray diffraction and electron microscopy. Poorly-crystalline hydroxyapatite (HAP: Ca(5)(PO(4))(3)OH) formed in control solutions over the experiment period of 24 h, following a second-order dependence on P concentration. Humic acid and Mg(2+) significantly inhibited formation of HAP, allowing formation of a more soluble amorphous Ca phosphate phase (ACP), and thus reducing the precipitation rate constants by 94-96%. Inhibition caused by Mg(2+) results from its incorporation into Ca phosphate precipitates, preventing formation of a well-crystalline phase. Humic acid likely suppressed Ca phosphate precipitation by adsorbing onto the newly-formed nuclei. Presence of oxalic acid resulted in almost complete inhibition of HAP precipitation due to preemptive Ca-oxalate formation. Carbonate substituted for phosphate, decreasing the crystallinity of HAP and thus reducing precipitation rate constant by 44%. Sulfate and Si-rich solids had less impact on formation of HAP; while they reduced precipitation in the early stage, they did not differ from the control after 24 h. Results indicate that components (e.g., Mg(2+), humic acid) producing relatively soluble ACP are more likely to reduce P stability and precipitation rate of Ca phosphate in soils and sediments than are components (e.g., SO(4)(2-), Si) that have less effect on the crystallinity.

The Inhibition of Calcium Phosphate Precipitation by Fetuin Is Accompanied by the Formation of a Fetuin-Mineral Complex

The present studies show that the previously reported ability of fetuin to inhibit the precipitation of hydroxyapatite from supersaturated solutions of calcium and phosphate in vitro is accompanied by the formation of the fetuin-mineral complex, a high molecular mass complex of calcium phosphate mineral and the proteins fetuin and matrix Gla protein that was initially discovered in the serum of rats treated with etidronate and that appears to play a critical role in inhibiting calcification in vivo. Rat serum potently inhibited the precipitation of calcium phosphate mineral when the concentration of calcium and phosphate were increased by 10 mm each, and the modified serum was incubated at 37 degrees C for 9 days; in the absence of serum, precipitation occurred in seconds. Large amounts of the fetuin-mineral complex were generated in the first 3 h of this incubation and remained throughout the 9-day incubation. Purified bovine fetuin inhibited the precipitation of mineral for over 14 days in a solution containing 5 mM calcium and phosphate at pH 7.4 at 22 degrees C, whereas precipitation occurred in minutes without fetuin. There was a biphasic drop in ionic calcium in the fetuin solution, however, from 5 to 3 mM in the first hour and from 3 to 0.9 mM between 20 and 24 h; these changes in ionic calcium are due to the formation of complexes of calcium, phosphate, and fetuin. The complex found at 24 h to 14 days is identical to the fetuin-mineral complex found in the serum of etidronate-treated rats, whereas the complex found between 1 and 20 h is less stable.

Inhibition of calcium phosphate precipitation by bile salts: a test of the Ca(2+)-buffering hypothesis.

The ability of bile salts to inhibit the precipitation of either calcium hydroxyapatite or its precursor, amorphous calcium phosphate, by reducing Ca2+ activity or poisoning nascent crystals was determined. When apatite precipitated rapidly (1-4 h), glycocholate and taurine-conjugated bile salts (up to 100 mM) had little effect on apatite formation, but prevented amorphous calcium phosphate precipitation by lowering Ca2+ activity. In contrast, glycodeoxycholate and glycochenodeoxycholate (2-3 mM) inhibited apatite formation for at least 24 h by poisoning embryonic apatite. When apatite precipitated slowly (> 24 h), all the dihydroxy bile salts prevented apatite formation for at least 4 days. At constant initial supersaturation, the phosphate concentration determined the degree of inhibition caused by the six bile salts mixed together in physiologic proportion. At low phosphate concentrations (1.2 mM) total inhibition was achieved by poisoning embryos (approximately -5 mM total bile salt), but with 4.0 mM phosphate only approximately 60% inhibition was attained (150 mM bile salt) by a combination of poisoning and Ca(2+)-buffering. Thus, at low supersaturation all dihydroxy bile salts can prevent apatite formation by reducing free Ca2+ (taurine and glycine conjugates) or poisoning embryos (glycine conjugates). With mixtures of bile salts at higher supersaturation, inhibition of apatite depends on a combination of poisoning and reduction of free Ca2+, mainly caused by glycodeoxycholate and glycochenodeoxycholate.

Inhibition of calcium phosphate precipitation by human salivary statherin: structure-activity relationships.

Previous studies of human statherin showed the active region for inhibition of secondary calcium phosphate precipitation (crystal growth) to reside in the highly charged amino-terminal one-third of this molecule, and the neutral tyrosine-, glutamine- and proline-rich carboxy-terminal two-thirds of the molecule is required for maximal inhibition of primary (spontaneous) precipitation. The purpose of the present study was to define more clearly the activities of these different molecular segments of statherin with respect to the two kinds of inhibitory activities. Peptides from statherin were prepared by specific proteolysis using trypsin, endoproteinase Arg-C, and activated factor X to produce the amino-terminal hexa-, nona- and decapeptides, respectively, and carboxypeptidase-A was used to obtain a peptide extending from residue 1 to about residues 32-37. The peptides were purified by anion exchange and gel filtration chromatography, and characterized and quantified by amino-acid analysis. Serially diluted samples of statherin and derived peptides were assayed to determine the concentrations, giving a standard 50% inhibition of precipitation (C50%) in assay systems designed for this purpose using polyaspartate as a standard. Results are expressed as (C50% statherin)/(C50% peptide). For inhibition of primary precipitation, these values were peptide(1-6), 0.20; peptide(1-9), 0.15; peptide(1-31/35), 0.24. For inhibition of secondary precipitation, the values were peptide(1-6), 3.8; peptide(1-9), 2.8; peptide(1-10), 1.9; peptide(1-32/37), 1.5. These quantitative findings show that maximum inhibition of primary precipitation by statherin requires the entire molecule. Thus, removal of a relatively small segment of its carboxy-terminal region results in a substantial reduction in inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Silica-induced Precipitation of Calcium Phosphate in the Presence of Inhibitors of Hydroxyapatite Formation

The promotion and the inhibition of hydroxyapatite formation by various substances were determined by measurement of the induction time of spontaneous precipitation (ti) from supersaturated solutions. Silica was found to decrease ti in Hepes-buffered (pH 7.2) supersaturated solutions with a wide range of calcium-to-phosphate ratios and concentrations. Also, in suspensions of the oral bacteria S. mutans or C. matruchotii in 1 mmol/L calcium, 7.5 mmol/L phosphate, and 50 mmol/L Hepes (pH 7.2), silica was capable of stimulating precipitation. Macromolecules derived from these bacteria by freezing and thawing appeared to be strong inhibitors of calcium phosphate precipitation. In the presence of silica, the effects of these bacterial inhibitors could be partially overcome, which supports the idea that silica in dental plaque is a promoter of calculus formation. In contrast, inhibition of calcium phosphate precipitation by a low-molecular-weight inhibitor, pyrophosphate, could not be counteracted by silica.

Proteoglycan inhibition of calcium phosphate precipitation in liposomal suspensions.

The major proteoglycan in cartilage (aggrecan) is a complex macromolecule with numerous chondroitin sulphate, keratan sulphate, and oligosaccharide substituents. It has been proposed that this macromolecule has an important role in regulating mineralization in this tissue, a process which is initiated by the deposition of apatite in matrix vesicles. We have used a liposome-centred endogenous precipitation method as a model for matrix vesicle mineralization to study the effect of the rat chondrosarcoma aggrecan and its chondroitin sulphate and core protein components on apatite formation from solution. Precipitation was initiated by encapsulating buffered (pH 7.4) 50 mmol/l KH2PO4 solutions in the aqueous centres of 7:2:1 phosphatidylcholine:dicetylphosphate:cholesterol liposomes, adding 2.25-2.65 mmol/l Ca2+ and 1.5 mmol/l total inorganic phosphate (PO4) to the suspending medium (pH 7.4, 22 degrees C), then making the intervening lipid membranes permeable to the Ca2+ ions with the calcium ionophore X-537A. Aggrecan (0.5%) in the suspending medium had no effect on intraliposomal precipitation, but severely reduced (approximately 70% reduction at 24 h) its subsequent spread into the medium. The chondroitin sulphate and core protein were similarly inhibitory. The degree to which aggrecan and its constituent parts inhibited precipitation correlated with their capacity to bind Ca2+ ions. These findings suggest that functional groups in aggrecan blocked apatite growth by linking via Ca2+ bridges to growth sites on the crystal surfaces. Similar Ca-mediated interactions may well have a critical regulatory role in cartilage mineralization.

Inhibition of calcium phosphate precipitation by human salivary acidic proline-rich proteins: structure-activity relationships.

Absence of precipitation of calcium phosphate salts onto tooth surfaces from human saliva, which is supersaturated with respect to calcium phosphate salts, has been attributed in part to the presence in the salivary secretions of a group of acidic proline-rich phosphoproteins (PRP). These macromolecules are considered to act by adsorbing onto dental enamel where they inhibit surface-induced precipitation of calcium phosphate salts. The inhibitory activity is known to be associated primarily with the amino-terminal region of the PRP. The aim of this study was to determine the features of the primary structure of this molecular segment responsible for inhibitory activity. The 30-residue, amino-terminal segment of PRP-3, which contains the two phosphoserines and 11 of the 13 carboxyl groups present in PRP-3, was obtained by tryptic digestion. This peptide, designated PRP-3(TI), was treated with thermolysin to give the monophosphopeptides, Val-PSer-Gln-Glu-Asp-Val-Pro and Leu-Val-Ile-Ser-Asp-Gly-Gly-Asp-PSer-Glu-Gln, and with alkaline phosphatase to give the dephosphorylated analog, PRP-3(TI)DP. The inhibitory activities of PRP-3(TI) and the derived peptides, a synthetic peptide, phosphoseryl-phosphoserine (PSer-PSer), and O-phosphoserine (PSer), were determined using an assay based on inhibition of seeded precipitation of calcium phosphate. Inhibitory activities, expressed as concentrations of inhibitors required to give standard inhibitory activities, were PRP-3(TI), 0.59 microM; PSer-PSer, 3.5 microM; the two monophosphopeptides, 29 and 32.5 microM; PRP-3(TI)DP, 56 microM; PSer, 329 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a protease derived from the oral bacterium Bacteroides loescheii on the inhibition of calcium-phosphate precipitation by human parotid saliva

A protease from the culture fluid of this microorganism was purified and characterized using DEAE-Sephadex and chromatofocusing and classified as a thiol protease with a mol. wt estimated as 27,500. It abolished the inhibitory activity of parotid saliva on the precipitation of calcium phosphate from supersaturated solutions. Such proteases may contribute to dental calculus formation.

Relationship between concentration of human salivary statherin and inhibition of calcium phosphate precipitation in stimulated human parotid saliva.

Human salivary secretions are supersaturated with respect to the calcium phosphate salts which form dental enamel, a property which provides important protection for the teeth. We previously proposed that statherin, a 43-residue phosphopeptide, plays a key role in this protective system by inhibiting or delaying potentially harmful precipitation of calcium phosphate salts in the salivary glands and mouth. The purpose of the present study was to determine if the concentrations of statherin in saliva, despite their wide normal range, are high enough to fulfill this function. Concentrations of statherin in stimulated human parotid saliva samples from 36 female and 32 male subjects, aged from 17 to 30 years, were determined by a single radial immunodiffusion method. Values found ranged from 3.0 to greater than 27.3 microM, with a mean value of 12.8 (S.D. +/- 5.46) microM. At concentrations below these values, statherin inhibited spontaneous precipitation of calcium phosphate salts from an assay system which was more supersaturated with respect to dicalcium phosphate dihydrate, and comparably supersaturated with respect to hydroxyapatite, than were human saliva samples. The inhibitory activities of five of the 65 stimulated parotid saliva samples assayed were greater than would be anticipated from their statherin concentrations. This unexplained discrepancy is not associated with the presence of the acidic proline-rich proteins in saliva, although these proteins also affect calcium phosphate precipitation. The results of this study show that statherin is present in stimulated human parotid saliva at concentrations and levels of activity which are consistent with its proposed biological function, and support the proposal that statherin plays a significant role in a system which provides a protective and reparative but stable environment for the teeth.

Structure-activity relationships of various bisphosphonates.

A variety of bisphosphonates with aliphatic side chains of increasing length, as well as 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP, formerly APD), dichloromethylene-bisphosphonate (Cl2MBP, formerly Cl2MDP), and dibromomethylene bisphosphonate (Br2MBP, formerly Br2MDP), were compared in vitro and in vivo to find (a) a possible relationship between structure and activity in order to give some indication about their mechanism(s) of action on bone and (b) the most efficient and safe compound having an effect on bone resorption. Some relationship was found between inhibition of calcium phosphate precipitation in vitro and of mineralization in vivo. No correlation existed, however, between any parameter measured and bone resorption. The number of calvaria cells in culture was decreased by compounds with a chain length greater than 5-C, by AHPrBP, Cl2MBP, and Br2MBP. Lactate production by these cells in vitro was increased by the long chain bisphosphonates and AHPrBP, and was decreased by Cl2MBP. No good correlation existed between the inhibition of bone resorption measured in vitro on calvaria and that seen in vivo on rat tibiae metaphyses. The latter was inhibited the most efficiently by the bisphosphonates longer than 5-C and by AHPrBP; these were 10 times more effective than Cl2MBP. Taking into account all factors, 1-hydroxypentylidene-1,1-bisphosphonate and AHPrBP seem to be the most active compounds to inhibit bone resorption.

Effect of human salivary proteins on the precipitation kinetics of calcium phosphate.

Inhibition of calcium phosphate precipitation in saliva, and prevention of the formation of mineral accretions on tooth surfaces, has been ascribed to the existence of inhibiting salivary macromolecules. Marked reductions in the crystal growth rate of hydroxyapatite (HA) seeds were measured in supersaturated solutions containing either of two proline-rich proteins, PRP1 or PRP3, or statherin; the three macromolecules were isolated from human parotid saliva. The reductions were also observed when the HA seeds were pretreated with solutions of the macromolecules before adding them to the supersaturated calcium phosphate solution. This effect was very similar in the case of the two PRPs and it was directly related to the extent of adsorption site coverage of these proteins on the HA seeds. The effect of statherin was larger than anticipated from its adsorption behavior. However, comparison on the basis of number of moles adsorbed per unit area of HA shows that the PRP are more effective inhibitors than statherin. The macromolecule concentrations used were considerably lower than those in the salivary secretions, therefore these macromolecules could readily prevent mineral accretion on tooth surfaces through their adsorption onto the enamel surface.

Inhibition of calcium phosphate precipitation by human salivary secretions

Although human saliva is supersaturated with respect to most basic calcium phosphate salts, it lacks the properties which are characteristic of metastable solutions. The purpose of this investigation was to elucidate the reasons for this anomaly. Saliva, salivary ultrafiltrates and the protein fraction of saliva were examined for their effect on the precipitation of calcium phosphate from supersaturated solutions. Salivary ultrafiltrates had little or no effect, but saliva and its macromolecular fraction strongly inhibited precipitation. Serum proteins were inactive, even at 50 times active salivary protein concentrations. The specific inhibitory activity, per unit of protein, of the parotid saliva from 15 subjects varied by a factor of three. Comparison of the unstimulated and stimulated parotid and submandibular secretions of one subject showed activity to be present in the unstimulated salivas and to increase substantially in the stimulated secretions.