TGase Cross-linking Research Articles

Effect of ultrasound on the structure and functional properties of transglutaminase-crosslinked whey protein isolate exposed to prior heat treatment

Effects of ultrasound treatment (20 kHz, 41–45 W cm−2, 0, 20, 40 or 60 min) on the physicochemical, functional properties and elements of the secondary structure of transglutaminase (TGase)-crosslinked whey protein isolate (WPI) exposed to prior thermal treatment (75 °C, 15 min) were investigated. The largest molecular size of proteins in the TGase-crosslinked WPI was observed after the ultrasound and thermal pre-treatment (HU-WPI-TGase). HU-WPI-TGase had the maximum intrinsic fluorescence intensity, with highest loss of free amino groups. Ultrasound-treated WPI (U-WPI) showed more (13%) emulsifying activity and more (63%) foaming ability than untreated WPI, but HU-WPI-TGase had higher foam stability and lower emulsifying activity than U-WPI. FTIR analysis indicated that ultrasound, heat treatment and TGase cross-linking had effects on the β-sheet, β-turn and random coil of WPI. The outcomes from this study show a potential application in providing novel functional ingredients for the dairy industry.

Cross-linking and film-forming properties of transglutaminase-modified collagen fibers tailored by denaturation temperature

Transglutaminase (TGase) catalyzing crosslinking between intra- and inter-chain glutamine and lysine peptide residues has been used for modifying protein’s structure. However, its enzymatic performance on albuminoids such as collagen was still not completely illustrated. Herein, we investigated the crosslinking efficiency of films preheated at different temperatures and estimated its physicochemical properties. The electrophoresis results showed that the extracted collagen had typical triple helix structure but reduced and even disappeared as temperature increased. Accordingly, X-ray diffraction (XRD) indicated that the amount of triple helices decreased, corresponding to the decreases in thermal stability and mechanical properties of films. TGase crosslinking decreased the thickness of all films, while mechanical properties and thermal stability had a significant improvement especially at 45 °C and 65 °C. With the proper equilibrium of denature temperature and TGase crosslinking, the tailored film-forming properties of collagen can offer a potential to engineer collagenic material for biodegradable and edible packaging applications.

Effects of proteolysis and transglutaminase crosslinking on physicochemical characteristics of walnut protein isolate

In order to improve the functional properties of walnut protein isolate (WPI), the effect of partial hydrolysis with trypsin, crosslinking with transglutaminase (TGase), and combinations of both treatments on the physicochemical characteristics of WPI was investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that WPI could be easily digested into smaller peptides by trypsin and could also be crosslinked into aggregates with high molecular weight. Hydrolyzing crosslinked WPI significantly increased its solubility from 9.73% to 54.54%. Scanning electron microscopy indicated that treatment with trypsin or TGase alone had a notable effect whereas the mixture of enzyme and TGase had a relatively lower effect on the microstructure of WPI. Hydrolysis did not affect the sulfhydryl (-SH) content while TGase crosslinking significantly reduced (-SH) content from 92.9 μmoL/g to 47.06 μmoL/g. Hydrolysis markedly increased the surface hydrophobicity (H0), while crosslinking did not significantly change H0. TGase treatment increased the viscosity of hydrolysates, but the viscosity of trypsin hydrolysates was not altered. Treatment of WPI with both TGase and trypsin showed the lowest initial G′ and G″ values due to the highest solubility. Thus, WPI with proteolysis and transglutaminase treatment has great potential for applications in the food industry.

Transglutaminase-set colloidal properties of wheat gluten with ultrasound pretreatments

The low solubility of wheat gluten limits its accessibility. This work aimed to study the impact of ultrasonic pretreatments on the gelation of wheat gluten. The pretreatments included ultrasound combined with alkali, urea, Na2SO3, with or without the addition of transglutaminase (TGase). The gel strength of wheat gluten was 287g/cm2 after treatment with Na2SO3/ultrasound/TGase. The free sulfhydryl and disulfide bond content was significantly affected by ultrasound treatment. After treatments including TGase crosslinking, the molecular weight of wheat gluten complexes became larger. The network formed by the wheat gluten was transformed into a dense and homogenous structure after the pretreatment with Na2SO3/ultrasound/TGase. The content of random coil of wheat gluten increased. The gelation of wheat gluten could also be significantly enhanced by Na2SO3/ultrasound treatment followed by TGase treatment. Using physical and chemical pretreatments to allow TGase to enhance the gelation of wheat gluten may increase its uses as a food additive.

Improved thermal-stability and mechanical properties of type I collagen by crosslinking with casein, keratin and soy protein isolate using transglutaminase

The inferior thermal- stability of collagen hinders its extensive application in food industry, including edible packaging. To improve the thermal- stability and mechanical properties of collagen, we attempted to crosslink collagen with some proteins possessing excellent thermal stability (i. e., casein, keratin and soy protein isolate (SPI)). Observed from the SDS- PAGE and particle size distribution, some complexes with higher molecule weight and relative bigger size particle occurred in the protein mixture, especially after TGase crosslinking. Importantly, the crosslinking greatly improved the thermal- stable property of protein complex, especially that of the collagen- casein complex judged from differential scanning calorimetric (DSC). Moreover, the crosslinking enhanced the mechanical properties of the combined films in terms of tensile strength (TS) and elongation at break (EAB). Also, some obvious differences in morphology of proteins before and after TGase crosslinking were observed by scanning electron microscopy (SEM). These impacts of TGase crosslinking with heat- resistant proteins on collagen features were associated with the conformational changes of the protein complex analyzed by Fourier transform infrared spectroscopy (FTIR). In conclusion, TGase crosslinking with higher thermally stable proteins could be an effective method to contribute to collagen’ application in food packaging field.

Effect of transglutaminase cross-linking on the conformational and emulsifying properties of peanut arachin and conarachin fractions

Peanut protein is widely consumed in many countries of the world. The effects of transglutaminase (TGase)-induced cross-linking on the functional properties of peanut protein fractions of arachin and conarachin were investigated. TGase promoted protein cross-linking and caused lower protein solubility. SDS-PAGE and high-performance size exclusion chromatography displayed that larger polymers or aggregates were formed during TGase treatment and the conarachin fraction was more susceptible to cross-linking than arachin. Conarachin has higher surface hydrophobicity (H0) and emulsifying properties than arachin. TGase incubation led to decrease in total free SH content and increase in exposed SH and H0 before 90 min treatment, suggesting unfolding of the protein molecules. The emulsifying properties for conarachin were also progressively improved by TGase treatment before 90 min, which was consistent with the change of H0. This study presented the variance in structure and emulsifying properties changes between arachin and conarachin fractions and their functional properties change caused by TGase cross-linking.

Textural performance of crosslinked or reduced-calcium milk protein ingredients in model high-protein nutrition bars

Transglutaminase (Tgase) crosslinking and calcium reduction were investigated as ways to improve the texture and storage stability of high-protein nutrition (HPN) bars formulated with milk protein concentrate (MPC) and micellar casein concentrate (MCC). The MPC and MCC crosslinked at none, low, and high levels, and a reduced-calcium MPC (RCMPC) were each formulated into model HPN bars. Hardness, crumbliness, moisture content, pH, color, and water activity of the HPN bars were measured during accelerated storage. The HPN bars prepared with MPC were harder and more cohesive than those prepared with MCC. Higher levels of Tgase crosslinking improved HPN bar cohesiveness and decreased hardening during storage. The RCMPC produced softer, yet crumblier HPN bars. Small textural differences were observed for the HPN bars formulated with the transglutaminase crosslinked proteins or RCMPC when compared with their respective controls. However, modification only slightly improved protein ingredient ability to slow hardening while balancing cohesion and likely requires further improvement for increased applicability in soft-texture HPN bars.

Transglutaminase and high pressure effects on heat-induced gelation of Alaska pollock (Theragra chalcogramma) surimi

A prior high pressure processing (HPP) treatment has previously been shown to enhance subsequent (at atmospheric pressure) low temperature ‘setting’ (glutamyl-lysine, non-disulfide protein crosslinking) induced by transglutaminase (TGase; endogenous or added microbial enzyme). This enhanced setting induces greater gel strength and deformability of subsequently cooked surimi gels. We sought to determine if 25°C incubation carried out simultaneous with, rather than following, HPP might similarly induce a setting effect on subsequently cooked gels. Pollock surimi pastes (± added microbial TGase) were subjected to HPP 200, 300, or 400MPa; at 5°C (little or no setting effect expected during HPP at this temperature) for 30min or at 25°C (optimal setting temperature for pollock pastes) for 30, 60, or 120min. Pastes were then directly cooked (90°C for 20min) (pressureP°C/time>C), or first allowed to undergo setting at atmospheric pressure (25°C for 30, 60, or 120min), followed by cooking (90°C 20min) (pressureP°C/time>Stime>C). With no microbial TGase added to the raw paste (e.g., endogenous TGase only), 300MPaP5°C/30min>S25°C/120min>C induced highest gel fracture stress and strain. The same treatment but with HPP at 200 or 400MPa gave only slightly lower fracture stress (gel strength). Increasing the temperature of the HPP treatment to 25°C (300MPaP25°C/30min>S25°C/120min>C), even with longer HPP time (300MPaP25°C/60 or 120min>C), gave weaker gels, similar to those obtained by setting and cooking without a prior HPP treatment (S25°C/60min>C). Thus, attempting to induce TGase crosslinking by setting at 25°C during HPP treatment actually seemed detrimental to gel strength development. However, when HPP was carried out at 25°C and microbial TGase was added, gel strength and deformability (fracture stress, strain) were enhanced above that of all other treatments tested. All treatments containing microbial TGase evidenced enhanced protein polymerization. Scanning electron microscopy revealed a more dense and fibrous structure in such gels, and reduction of free thiol (SH) groups was noted as a result of microbial TGase addition.

Monitoring the effect of high pressure and transglutaminase treatment of milk on the evolution of flavour compounds during lactic acid fermentation using PTR-ToF-MS

In this study, the effects of thermal or high hydrostatic pressure (HHP) treatment of a milk base in the absence or presence of a transglutaminase (TGase) protein cross-linking step on the flavour development of yoghurt were investigated. The presence of several tentatively identified volatile flavour compounds (VOCs), both during the enzymatic treatment and the lactic acid fermentation of the milk base, were monitored using a proton transfer reaction time-of-flight mass spectrometer (PTR-ToF-MS). The formation of the major flavour compounds (acetaldehyde, diacetyl, acetoin, and 2-butanone) followed a sigmoidal trend described by the modified Gompertz model. The HHP treatment of milk increased significantly the volatile compound formation rate whereas it did not affect the duration of the lag phase of formation, with the exception of acetaldehyde and diacetyl formation. On the contrary, the TGase cross-linking of milk did not significantly modify the formation rate of the volatile compounds but shortened the duration of the lag phase of their formation.

Physicochemical properties and residual antigenicity of transglutaminase cross-linked sodium caseinate hydrolysates

Transglutaminase (TGase) cross-linked sodium caseinate (NaCN) was hydrolysed with Alcalase, Corolase 7089, Promod 144MG (papain) and Corolase PP. NaCN hydrolysates were also generated with these food-grade proteolytic preparations and subsequently cross-linked. The contribution of the order of partial TGase cross-linking and proteolysis to the properties of the resultant NaCN hydrolysates was studied. At essentially similar degree of hydrolysis (DH) values, the order of cross-linking and hydrolysis did not appear to affect the physicochemical (reversed-phase and gel permeation) profiles of the hydrolysates. Furthermore, TGase cross-linking, and the order of enzymatic modification (cross-linking/hydrolysis) did not significantly ( P > 0.05) alter the residual antigenicity properties of NaCN or of high DH (>18%) hydrolysates. Under the experimental conditions, it was not possible to determine if cross-linking affected the extent of hydrolysis. Overall, these results indicate that DH was considerably more important than cross-linking in determining the physicochemical and residual antigenicity properties of the hydrolysates.

Molecular and structural characteristics of cod gelatin films modified with EDC and TGase

Cod gelatin films before and after cross-linking of gelatin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or transglutaminase (TGase) have been characterized by Fourier transform infrared (FT-IR) spectroscopy and differential scanning calorimetry (DSC) analysis. For comparison, a film prepared from unmodified pig gelatin has been also analysed. The difference spectra showed that cod gelatin during the film formation involved first of all water-to-amide hydrogen bonds, and the film from pig gelatin contained water-to-amide, amide-to-amide and water-to-water hydrogen bonds. A higher number of hydrogen bonds in the structure of the film from pig gelatin contributed to much better recovering of the helical structure in this film than in the film from cod gelatin, as the peaks at about 1663 cm −1 in the amide I band and at about 1537 cm −1 in the amide II band in the second-derivative spectra revealed. The recovered helical structure, in turn, resulted in a significantly higher melting enthalpy value in the case of the film from the pig gelatin. After modification of cod gelatin with EDC or TGase, the inter-chain cross-linkages formed in the films led to the conformation of gelatin with no indications of helical ordering. An increase of melting temperature of gelatin films by 7 °C on EDC and by 10 °C on the TGase modifications was related to the formation of covalent cross-links, and a decrease of glass temperature by 28 °C and 7 °C on EDC and TGase cross-linking, respectively, demonstrated the plasticizing effect of water.

Effects of Microfluidization Treatment and Transglutaminase Cross-Linking on Physicochemical, Functional, and Conformational Properties of Peanut Protein Isolate

Peanut protein isolate (PPI) was treated by high-pressure microfluidization (40, 80, 120, and 160 MPa) and/or transglutaminase (TGase) cross-linking. It was found that individual microfluidization at 120 MPa was more effective in improving the solubility, emulsifying properties, and surface hydrophobicity of PPI than at other pressures (e.g., 40, 80, or 160 MPa). Individual TGase cross-linking also effectively changed the physicochemical and functional properties of PPI. Microfluidization (120 MPa) or TGase cross-linking caused the unfolding of PPI structure, resulting in the decrease of α-helix and β-turns levels and the increase of β-sheet and random coil levels, as proved by Fourier transform infrared (FTIR) and circular dichroism (CD) spectra. Compared with individual treatments, microfluidization followed by TGase cross-linking significantly (p < 0.05) improved the emulsion stability during long-term storage (20 days). Moreover, the combined treatments led to looser structure of PPI and resulted in more obvious changes in physicochemical properties.

Emulsifying properties of the transglutaminase-treated crosslinked product between peanut protein and fish (Decapterus maruadsi) protein hydrolysates

Defatted peanut meal, a protein-rich by-product from the oil extraction industry, is underutilised owing to its inferior functional properties. In this study, transglutaminase (TGase) crosslinking and proteolysis were used to improve the emulsifying properties of peanut protein isolate (PPI) extracted from the meal. PPI and PPI hydrolysate (PPIH) were conjugated separately with fish (Decapterus maruadsi) protein hydrolysate (DPH), catalysed by TGase to obtain improvements in the emulsifying properties. Analyses by electrophoresis and high-performance liquid chromatography indicated that polymers were formed in all TGase-treated samples. In emulsions of PPIH, PPI-DPH and PPIH-DPH the volume/surface average particle diameter (d(32) ), creaming and instability phenomenon were decreased and the zeta-potential was increased after TGase treatment, showing improved emulsifying activity and emulsion stability. In the case of PPI, TGase treatment had no effect on the emulsifying activity, but the emulsion stability of TGase-treated PPI was improved. The study showed that TGase crosslinking and proteolysis could improve the emulsifying properties of PPI, while proteolysis followed by TGase crosslinking proved more efficient. The emulsifying properties of the heterologous protein systems of PPI-DPH and PPIH-DPH were also improved by TGase treatment.

P4-160: Transglutaminase 1, Transglutaminase 2 and Transglutaminase-catalyzed cross-links colocalize with the pathological lesions in Alzheimer's disease brain

Alzheimer's disease (AD) is characterized by several pathological lesions, in particular senile plaques (SPs), cerebral amyloid angiopathy (CAA) and neurofibrillary tangles (NFTs), predominantly consisting of self-aggregated proteins amyloid-beta (Aβ) and tau, respectively. Transglutaminases (TGases) are inducible enzymes, capable of modifying the conformational and/or structural properties of proteins, such as Aβ and tau, by inducing intra- and intermolecular covalent cross-links. Both Aβ and tau are substrates for TGase cross-linking activity, which suggests that TGases play a role in the initiation and persistence of the aggregation process of both Aβ and tau in AD brains. The aim of this study was to investigate the association of Transglutaminase 1 (TG1), Transglutaminase 2 (TG2) and TG-catalyzed cross-links with the pathological lesions of AD. For this purpose, a panel of well-characterized antibodies against TG1, TG2 and the TG-catalyzed cross-links was used in immunohistochemistry. We observed the immunoreactivity for TG1, TG2 and TG-catalyzed cross-links in NFTs. In addition, both TG2 and TG-catalyzed cross-links colocalize with Aβ in SPs. Furthermore, staining of TG2 and TG-catalyzed antibodies was also observed in CAA, although specific colocalization with deposited Aβ in CAA was absent. We conclude that both TG1 and TG2 are present in SPs, CAA and NFTs, and that these enzymes display cross-linking activity in AD lesions. We suggest that both TG1 and TG2 are involved in the initiation and/or persistence of the protein aggregation processes underlying the formation of SPs and CAA, and NFTs in AD brain.

Modification of the nitrogen solubility properties of soy protein isolate following proteolysis and transglutaminase cross-linking

The effect of (a) limited hydrolysis [0.5–2.0% degree of hydrolysis (DH)] with Alcalase™, (b) cross-linking with transglutaminase (TGase) and (c) combinations of these modifications on the nitrogen solubility (pH 3–8) of soy protein isolate (SPI) was investigated. Between pH 3.0 and 5.0, SPI hydrolysates, hydrolysates of cross-linked SPI and the cross-linked products of SPI hydrolysates displayed significant ( P<0.05) increases in solubility compared to unmodified SPI. Cross-linking pre- or post hydrolysis did not alter the overall trend of increased ( P<0.05) solubility relative to the unmodified control at low pH. At 2% DH, cross-linking pre- or post-hydrolysis resulted in greater solubility ( P<0.05) than that observed in hydrolysates per se at low pH. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) indicated that the 22 kDa 11S basic polypeptide was relatively resistant to Alcalase hydrolysis and that the 18 and 22 kDa 11S basic polypeptides were not susceptible to TGase cross-linking. The results demonstrate that a combination of enzymatic treatments and the order in which they are applied may have potential for creating novel food ingredients with improved functional properties, especially those properties that are dependant on high solubility at low pH.

Effect of cross-linking with transglutaminase on the heat stability and some functional characteristics of sodium caseinate

The effect of heating (140 °C, 0–60 min between pH 6.0 and 7.0) on the turbidity, pH 4.6 soluble amino group content and urea PAGE profiles of sodium caseinate (NaCN) and transglutaminase (TGase)-treated NaCN was determined. pH-dependent heat-induced changes in the turbidity and urea PAGE profiles of NaCN were initially attributed to casein aggregation followed by subsequent degradation on extended heating. Cross-linked NaCN samples (incubated with TGase at 20 °C, [E:S] of 1:50 and 1:20 for 185 min) were generally less turbid and had lower pH 4.6 soluble amino group content on heating than unmodified NaCN. The nitrogen solubility of cross-linked NaCN was improved at pH 2.0, 3.0 and 5.0. Some improvements in emulsifying activity index and stability of cross-linked NaCN were observed at pH 5.0 and 10.0. The improved heat stability and nitrogen solubility observed after TGase cross-linking may help extend the range of applications for NaCN.

Physicochemical and nitrogen solubility properties of Bacillus proteinase hydrolysates of sodium caseinate incubated with transglutaminase pre- and post-hydrolysis.

Sodium caseinate (NaCN), hydrolyzed with Protamex, a Bacillus proteinase preparation, to 0.5, 1.3, and 17.5% degrees of hydrolysis, was incubated with transglutaminase (TGase) for 3, 42, and 290 min at enzyme/substrate ratios of 1, 1, and 10% (w/w), respectively, pre- and post-hydrolysis. The electrophoretic, reversed-phase high-performance liquid chromatography (RP-HPLC) and nitrogen solubility profiles of the modified products were investigated. Combinations of hydrolysis and incubation with TGase generated products displaying novel physicochemical and nitrogen solubility properties. Significant changes in sodium dodecyl sulfate (SDS) and urea polyacrylamide gel electrophoresis profiles were apparent in the modified caseinate samples. Extensive TGase cross-linking resulted in polymers that were unable to enter the resolving gel during SDS polyacrylamide gradient gel electrophoresis. Extensive combined enzymatic modification resulted in peptides eluting earlier on RP-HPLC than limited combined enzymatic modification or limited hydrolysis. Combination of enzymatic treatments resulted in significantly (P < 0.005) improved solubility around pH 4.6, compared to incubation with TGase or hydrolysis of NaCN alone.

Chapter 5 Analysis of protein transglutamylation in apoptosis

Publisher Summary This chapter focuses on protein isolation and characterization techniques, developed specially for Transglutaminase (TGase) research. Several of these techniques were invented and introduced for the analysis of keratinocyte-cornified envelope (CE), a specialized 15- to 20-nm-thick structure that is tightly cross-linked by three different TGases directly below the cell membrane of cornifying keratinocytes are discussed. The chapter describes tools to perform a systematic analysis of protein transglutamylation and cross-linking in the CE as well as appropriately chosen apoptotic cell culture systems. Cross-linked tissue proteins harbor inter- or intrachain ɛ-(γ-glutamyl)-lysine linkages between α-aminoacyl peptide chains. The determination of protein cross-link density within the tissue relies on the liberation of free ɛ-(γ-glutamyl)-lysine isodipeptide from larger protein structures by fragmentation in smaller peptides and exhaustive digestion by ectopeptidases that do not split the isopeptide bond. Because TGase cross-linking characteristically results in supracolloidal aggregates of proteins, as exemplified in the case of apoptotic bodies or keratinocytecornified envelopes, these tightly cross-linked macropolymeric structures often require freedom from the non-cross-linked proteins.

All-trans retinoic acid blocks the antiproliferative prodifferentiating actions of 1,25-dihydroxyvitamin D3 in normal human keratinocytes.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and all-trans retinoic acid (RA), the active metabolites of vitamins D and A respectively, regulate the proliferation and differentiation of keratinocytes. Both the vitamin D receptor (VDR) and the retinoic acid receptor family (RAR) bind to DNA response elements as heterodimers with the retinoic X receptor (RXR), suggesting that there are pathways of action that are shared by both compounds. Therefore, we examined the interactions of 1,25(OH)2D3 and RA upon the proliferation and differentiation of normal human keratinocytes (NHK) and of a squamous cell carcinoma cell line, SCC4. Although both 1,25(OH)2D3 and RA were each able to inhibit NHK proliferation in a dose-dependent manner, when they were administered in combination, proliferation was stimulated, suggesting mutual antagonism. In contrast, SCC4 cells proved insensitive in terms of proliferation to 1,25(OH)2D3 and to all but the highest concentration (10(-6) M) of RA. 1,25(OH)2D3 exerted a biphasic effect on transglutaminase (TGase) and involucrin (INV) mRNA levels, with maximal stimulation at 10(-9) M. RA inhibited TGase and INV mRNA levels and antagonized the stimulation by 1,25(OH)2D3. A similar pattern was observed for TGase protein, but, RA, which, by itself, reduced INV, markedly enhanced the ability of 1,25(OH)2D3 to raise INV levels, possibly by inhibiting 1,25(OH)2D3-stimulated TGase activity and cross-linking of soluble INV into the insoluble cornified envelope (CE). Thus, in NHK cells, RA antagonizes the antiproliferative prodifferentiating actions of 1,25(OH)2D3, but assessment of a single marker, such as INV protein, may be misleading.

Molecular cloning of mouse erythrocyte protein 4.2: a membrane protein with strong homology with the transglutaminase supergene family.

We report the molecular cloning and characterization of mouse erythrocyte protein 4.2 (P4.2). Mouse erythrocyte P4.2 is a 691-amino-acid protein with a predicted MW of 77 kDa. Northern blot analysis detected a 2.2-kb transcript in mouse reticulocytes, compared with a 2.4- to 2.5-kb transcript in human reticulocytes, which is consistent with the absence of the 30-amino-acid splicing insert in mouse erythrocyte P4.2 that is found in the human protein (isoform I). Like the human erythrocyte P4.2, mouse erythrocyte P4.2 contains regions strikingly homologous with the transglutaminase (TGase) proteins although it too most likely lacks TGase crosslinking activity. Mouse P4.2 is on average 73% identical with human erythrocyte P4.2, although regional variations exist, with greatest conservation in the regions of the molecule that contain the TGase active site, the TGase calcium-binding site, and a band 3 binding site. Hydropathy analysis reveals a protein containing a series of hydrophobic domains, similar to the situation for human P4.2 and consistent with its tight binding to the membrane, although the mouse P4.2 is missing both the strongly hydrophilic region and adjacent highly charged region that are present in the human protein, suggesting that the two proteins could differ in their physical characteristics, binding associations, or functional properties. The availability of the complete mouse erythrocyte P4.2 cDNA should help in the design of P4.2-deficient animal models (for example, ribozyme or homologous recombinant "knockout" models) that should accelerate the understanding of P4.2 function in both erythroid and non-erythroid cells.