Proteins are essential macromolecules that play diverse roles in the physiology and health of horses. They are composed of amino acids and are involved in various biological processes, including tissue growth, repair, and the synthesis of enzymes and hormones. Dietary proteins are a key component of equine nutrition, influencing muscle development, immune function, and overall performance. Horses require a balanced intake of essential amino acids, which must be obtained through their diet, as they cannot be synthesized endogenously. This page compiles peer-reviewed research studies and scholarly articles that explore the types, functions, and dietary requirements of proteins in horses, as well as their impact on equine health and performance.
Ek N.A method for the rapid electrophoresis on a cellulose acetate membrane of serum proteins from horses, sheep and pigs is discussed. The various main globulin fractions in the serum of these animals were experimentally identified. Normal values for the percentage composition of serum from normal horses, sheep and pigs were calculated. In the horse there was great individual variation in the shape of the β-fraction, assumed to be due to different transferrin types. The mean value for β-globulin of 19.5 % in the horse was higher than for the other two species. The albumin percentage was highest ...
Van den Oord AH, Wesdorp JJ, Van Dam AF, Verheij JA.In commercial samples of equine myoglobin and samples of equine and bovine myoglobin prepared in the laboratory, a small amount of the protein was present as an aggregate. The presence of the myoglobin aggregate could be demonstrated by gel filtration on Sephadex G-100 Superfine, which also provided a means of isolating it. Gel filtration on Sephadex G-100 showed the molecular weights of the equine and bovine moyglobin aggregates to be about 35000 and 34000 respectively, thus supporting the hypothesis that they are dimers. This was confirmed for the equine myoglobin by ultracentrifugation meas...
Simon SR, Cantor CR.The UV circular-dichroism spectra of human and horse hemoglobins have been determined at various degrees of partial saturation with oxygen. Spectra of the two native hemoglobins were compared with spectra of the corresponding proteins modified with a reagent known to eliminate the conformational rearrangement normally associated with cooperativity. Such comparison indicates that one region, around 260 mmu, is sensitive chiefly to the state of the hemes; changes in another region, around 285 mmu, may be correlated with the conformational transformation linked to cooperative interactions. All ci...
Montgomery PC, Dorrington KJ, Rockey JH.Three independent methods have been used
to determine the molecular weights of the heavyand light-polypeptide chain subunits of equine yGab-,
yGc-, and yT-immunoglobulins. Extensively reduced
and alkylated proteins were filtered through standard
columns of Sephadex G-100 or G-200 in 8 M urea405
M propionic acid. Subunit molecular weights were obtained from the linear elution volume, V,, us. logarithm
molecular weight relationship defined for each column
with rabbit yG-globulin heavy and light chains and
horse heart cytochrome c. Molecular weights also were
determined by equilibrium se...
Williams MA, Harrison PM.Horse ferritin was fractionated both by starch-gel electrophoresis and by gel filtration on Sephadex G-200. Monomer fractions contained up to 98% of monomer and oligomer fractions up to 76% of oligomers as determined by quantitative electron microscopy. Percentages obtained from electron micrographs correlated well with analytical starch-gel electrophoretograms and ultracentrifuge patterns. Amino acid analyses of monomer- and oligomer-enriched fractions showed no significant differences. Ferritin oligomers did not apparently dissociate on dilution for electron microscopy or on storage. Apoferr...
Turner GA, Taylor DM.The interactions between tetravalent plutonium and horse serum proteins were studied in vitro by electrophoresis on cellulose acetate and by gel filtration. The results show that in horse serum, as in other mammalian sera, the plutonium is associated principally with the transferrin component of the beta1-globulins. The formation of the plutonium-transferrin complex requires the presence of HCO3-, and plutonium is displaced from the complex by excess iron, thus indicating that similar binding sites may be involved in the complexing of iron and plutonium. The plutonium complex is considered to ...
Babel I, Stella RC, Prado ES.Previous experiments indicated that horse urinary kallikrein (UK) hydrolyzes salminei- e and polyarginine, a but not polylysine. This paper reports the action of UK on bradykinyl-serine, methionyllysyl-bradykinin and lysyllysyl-bradykinin.
Carpenter MA, Broad TE.Transferrin, the iron transport protein of the blood, is highly polymorphic in many species, including the horse. A number of sequence polymorphisms that distinguish several of the variants of horse transferrin are reported here. Previous studies indicated that exons 12 and 15 were likely to be polymorphic. Sequencing regions of exons 12 and 15 from D and R variants revealed 10 nucleotide substitutions that encoded six amino acid replacements. The F1, F2, H2, and * variants were identical to D, and the O variant was almost identical to R, in the regions studied. The data indicated that the hor...
Caffrey CR, Ryan MF.An excretory-secretory (ES) preparation derived from adult Strongylus vulgaris in vitro was assessed for proteolytic activity using azocasein and synthetic, fluorogenic, peptide substrates. Fractionation was by molecular sieve fast protein liquid chromatography (molecular sieve FPLC) and resolution by gelatin-substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis (gelatin-substrate SDS-PAGE). The cysteine proteinase activator, dithiothreitol (DTT), enhanced azocaseinolysis and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NMec) by the ES pre...
Furr M, Chickering WR, Robertson J.To determine normal CSF electrophoresis patterns in horses, and to determine whether the electrophoretic scans from horses with cervical compression differ from those of neurologically normal horses. Methods: 32 horses assigned to 1 of 2 groups: neurologically normal (n = 18) or cervical compression (n = 14). Methods: CSF was collected from 18 neurologically normal horses referred to the Marion duPont Scott Equine Medical Center, and protein electrophoresis was performed to describe the normal equine CSF electrophoretogram. Results of CSF electrophoresis from 14 horses with cervical compressio...
Jalali GR, Rezai A, Underwood JL, Mowbray JF, Surridge SH, Allen WR, Matthias S.We have shown that most of the IgG present on term syncytiotrophoblast, membrane, microvesicles is bound to an 80 kDa protein antigen (R80K). Methods: Microvesicles were prepared from term human placenta, and the IgG eluted at pH3. Results: When IgG antibody was eluted at pH3 and reacted with acid-treated vesicles of other placentae, the alloantibody always bound to the preparation from which it was obtained, but only to about 10% of acid-treated preparations from other placentae. A similar polymorphic protein found in association with IgG antibody was found in term horse placentae. Cross-reac...
da Silva CMB, Cano FEM, Gaitskell-Phillips G, Vega FJP.Flow cytometry is a powerful tool for the analysis of cell samples formed of multipopulations, such as spermatozoa. In recent years, multiparametric cytometers have evolved, allowing the study of different cellular characteristics, such as protein expression, DNA analysis, or mitochondrial activity. Whether using traditional fluorescent dyes or fluorophore-conjugated antibodies, each cell or cellular component is individually stained, the sample is analyzed at high velocities, and then is displayed and interpreted in a dot-plot. We hereby describe the procedure to perform a multiparametric flo...
Böttiger LE.Erythrocyte sedimentation rate, total protein and fibrinogen, electrophoretic protein pattern, and total serum protein-bound carbohydrates have been determined in a number of domestic animals and compared to human values. The striking finding is that although the E.S.R. varies widely between various species, the fibrinogen content is of the same order of magnitude in all. The horse, which shows a very high E.S.R., has a well marked beta-globulin fraction as an outstanding feature, a finding that should be further studied. Blutsenkungsgeschwindigkeit, Gesamteiweiss und Fibrinogen, elektroforeti...
Atkinson JP.1. The globulins of both normal and diphtheria antitoxic serum exhibit chemically toward reagents the same reactions, being precipitated by magnesium sulphate and split up into fractions in precisely the same way. 2. All of the diphtheric antitoxic power of both normal and immunized serum is always carried by the globulin and its fractional precipitates. 3. During the fractional precipitation of the serum globulin of horses immunized from diphtheria toxin and horses not immunized from diphtheria toxin, some of the globulin is lost, likewise at the same time some of the antitoxic power of the g...
Braun J, Hochi S, Oguri N, Sato K, Torres-Boggino F.Three experiments were conducted to evaluate the effect of different macromolecule components (egg yolk, skim milk, and BSA) in a widely employed extender for cryopreservation of horse semen. Spermatozoal motility (MOT) and the percentage of spermatozoa with an intact plasma membrane (IPM) were evaluated in frozen-thawed samples. In the first experiment (four Draft Horse stallions, four ejaculates each) a standard freezing extender containing 20% whole egg yolk was modified by replacing extender components (glucose-EDTA solution, 11% lactose solution) with an increasing volume of a skim milk d...
The protein journalJanuary 18, 2023
Volume 42, Issue 1 24-36 doi: 10.1007/s10930-023-10092-x
Rodríguez MC, Mussio PE, Villarraza J, Tardivo MB, Antuña S, Fontana D, Ceaglio N, Prieto C.Equine chorionic gonadotropin (eCG) is a glycoprotein hormone widely used in timed artificial ovulation (TAI) and superovulation protocols to improve the reproductive performance in livestock. Until recently, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG). Here, a bioactive recombinant eCG (reCG) produced in suspension CHO-K1 cells was purified employing different chromatographic methods (hydrophobic interaction chromatography and reverse-phase (RP)-HPLC) and compared with a RP-HPLC-purifi...
Rousselle JC, Gillet G, Fillion G.The solubilization of the serotonergic 5HT1 and 5HT3 sites was performed with digitonin and sodium cholate at 1% (final concentration). Two binding sites for [3H]5HT were observed on rat or horse brain synaptosomal membranes solubilized with these detergents. The corresponding dissociation constants (KD) were 1-3 nM and 13-30 nM respectively. These values were closely similar to those corresponding to 5HT1 and 5HT3 sites located in intact membranes. The solubilized sites specifically bound 5HT. The effect of GTP decreasing the binding to 5HT1 sites was lost on solubilized 5HT1 sites; it was re...
Forsell PK, Lindberg A, Karlsson S, Lindgren JA, Claesson HE.It has been demonstrated that equine neutrophils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A(2) (cPLA(2)) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA(2) in equine neutrophils. To determine whether cPLA(2) from neutrophils was catalytically active, we purified the enzyme >6,500 fold with 3% recovery from equine neutrophils. The full-length cDNA sequence encoded a 749-amino acid protein. The deduced amino acid sequence...
Marchant EA, Semevolos SA.The marginal transitional zone is peripherally located within the diarthrodial joint, and represents the interface of articular cartilage, periosteum, and the fibrous joint capsule. The purpose of this study is to characterize the protein expression of matrix and molecular regulators in the marginal transitional zone of foals having osteochondrosis (OC) compared to normal foals. Several families of proteins with known roles in cartilage and bone development are investigated, including matrix molecules, Wnt signaling, apoptotic factors and paracrine cell signaling molecules. Our results demonst...
Fontes KFLP, Silva-Júnior LC, Nascimento SA, Chaves DP, Pinheiro-Júnior JW, Freitas AC, Castro RS, Jesus ALS.A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented e...
Alim S, Cheppali SK, Laitaoja M, Talluri TR, Jänis J, Swamy MJ.Fibronectin type-II (FnII) family proteins are the major proteins in many mammalian species including bull, horse and pig. In the present study, a major FnII protein has been identified and isolated from donkey (Equus hemionus) seminal plasma, which we refer to as Donkey Seminal Plasma protein-1 (DSP-1). The amino acid sequence determined by mass spectrometry and computational modeling studies revealed that DSP-1 is homologous to other mammalian seminal plasma proteins, including bovine PDC-109 (also known as BSP-A1/A2) and equine HSP-1/2. High-resolution LC-MS analysis indicated that the prot...
Kumar S, Malik P, Verma SK, Pal V, Gautam V, Mukhopadhyay C, Rai GP.Glanders, caused by the Gram-negative, nonmotile bacterium Burkholderia mallei, is a contagious and highly fatal disease of equines. During the last decade, the number of glanders outbreaks has increased steadily. The disease also has high zoonotic significance and B. mallei is listed biological warfare agent. The complement fixation test (CFT) is a routinely used and internationally recognized test to screen equine sera for the glanders. However, discrepant results have been observed using the CFT. The low sensitivity and specificity of the CFT and enzyme-linked immunosorbent assay (ELISA) ha...
Lind C, Chen J, Byrjalsen I.We developed a rapid, competitive enzyme immunoassay (EIA) for measuring 25-hydroxyvitamin D3 [25(OH)D3] in serum. The EIA was based upon 25(OH)D3-3-hemisuccinate covalently coupled to secondary amino groups grafted onto the polystyrene surface of microtiter wells. Optimal coupling conditions were established, and we found that inclusion of 40 mumol/L chloramine T, an agent not previously described for use in coupling to these plates, resulted in both more reproducible coupling as well as more than a twofold increase in the coupling efficiency. Before EIA, 25(OH)D3 was extracted from the serum...
Galbraith EA, McKellar QA.Flunixin was highly protein bound in the serum of dogs (92.2 per cent), goats (84.8 per cent) and horses (86.9 per cent). Meclofenamic acid was also highly protein bound, although there were larger differences between the extent of the binding in dogs (90.3 per cent), goats (84.7 per cent) and horses (99.8 per cent). Both flunixin and meclofenamic acid were potent inhibitors of the in vitro generation of thromboxane (Tx) B2 in blood. Flunixin inhibited the generation of TxB2 by 50 per cent of the maximum response (IC50) in dog, goat and horse blood at concentrations of 0.10, 0.02 and 0.04 micr...
Komori M, Higami A, Imai Y, Imaoka S, Funae Y.A form of P450 [termed P450(h-1)] was purified from the liver microsomes of a male horse to electrophoretic homogeneity. The specific content of the final P450(h-1) preparation was 14.8 nmol/mg of protein and the recovery was 0.38% of the microsomal P450. The apparent molecular weight of P450(h-1) was 52,000 Da. The absorption spectra of P450(h-1) indicated that P450(h-1) was a low- and high-spin mixed type P450 in the oxidized form. The reconstituted system containing P450(h-1) could catalyze benzphetamine N-demethylation, 7-ethoxycoumarin O-deethylation, and testosterone 16 alpha-hydroxylati...
Turki I, Hammami A, Kharmachi H, Mousli M.Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon' (Fd). scFv50AD1-Fd was expressed as soluble recombinant protei...
Marchal R, Caillaud M, Martoriati A, Gérard N, Mermillod P, Goudet G.The aim of this study was to investigate the role of growth hormone (GH) on in vitro cumulus expansion and oocyte maturation in equine and porcine cumulus-oocyte complexes (COCs), and to approach its way of action. Equine COCs were cultured in a control medium (TCM199, 5 mg/ml BSA, 1 microg/ml estradiol, and antibiotics) supplemented with either 0.5 microg/ml equine GH or 5 microg/ml equine LH. Porcine COCs were cultured in a basal medium (TCM199 with 570 microM cysteamine) supplemented with 0, 0.1, 0.5, or 1 microg/ml porcine GH or in a control medium (basal medium with 10 ng/ml epidermal gro...
Jimenez Rihuete P, Villarino N, Pelisiak A, Rubio-Martinez LM.Refractometric determination of total protein (TP) in synovial fluid (SF) is commonly used for diagnosis and monitoring of synovial sepsis in horses. Previous studies have shown that elevated concentrations of certain anticoagulants may overestimate refractometric determination of TP concentration. Objective: The aim of the study was to evaluate the effect of different concentrations of dipotassium EDTA (KEDTA) and lithium heparin (LH) on TP determination by using a hand-held refractometer in equine synovial fluid. Methods: Cross-section observational study. Methods: Thirty samples of synovial...
Butnev VY, Gotschall RR, Baker VL, Moore WT, Gout PW, Bousfield GR.Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structu...
Marti E, Wang X, Jambari NN, Rhyner C, Olzhausen J, Pérez-Barea JJ, Figueredo GP, Alcocer MJ.Insect bite hypersensitivity (IBH) is a seasonal recurrent skin allergy of horses caused by IgE-mediated reactions to allergens present in the saliva of biting insects of the genus Culicoides, and possibly also Simulium and Stomoxys species. In this work we show that protein microarrays containing complex extracts and pure proteins, including recombinant Culicoides allergens, can be used as a powerful technique for the diagnosis of IBH. Besides the obvious advantages such as general profiling and use of few microliters of samples, this microarray technique permits automation and allows the gen...
Leder L, Bosshard HR.It has long been known that antibodies to cytochrome c can distinguish between closely sequence-related cytochromes c. Because the 3-D-structure of the polypeptide chain is virtually identical among eukaryotic cytochromes c, antibody specificity is directed against amino acid substitutions within a common polypeptide folding pattern. The question arises if the specificity is observed at the level of the 3-D-structure (conformational epitopes) and/or at the level of the primary structure (sequential epitopes). Using rabbit sera to horse cytochrome c, we show that discrimination against the host...
Buffington CA, Knight DA, Kohn CW, Madigan JE, Scaman PA.The effects of 2 liquid formula diets differing in protein source were evaluated in orphan foals. The response of 7 foals fed a diet containing casein as the protein source, and 6 foals fed a diet containing a combination of whey and casein, was compared with the response in a reference group of 8 mare-raised foals. Orphaned foals were fed 150 kcal/kg of body weight/d, divided into 6 equal feedings of 25 kcal/kg. Formula intake was comparable among the experimental groups, and foals fed the liquid formula diet grew as well as mare-raised foals. There was no difference among groups in mean dail...
Singha H, Goyal SK, Malik P, Singh RK.Thermostabilizing effect of heavy water (D2O) or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water on in situ thermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of equine infectious anemia virus (EIAV) infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stabilit...
Smits K, Willems S, Van Steendam K, Van De Velde M, De Lange V, Ververs C, Roels K, Govaere J, Van Nieuwerburgh F, Peelman L, Deforce D, Van Soom A.During maternal recognition of pregnancy (MRP), a conceptus-derived signal leads to the persistence of the corpus luteum and the maintenance of gestation. In the horse, the nature of this signal remains to be elucidated. Several studies have focused on the changes in gene expression during MRP, but little information exists at the protein level. The aim of this study was to identify the proteins at the embryo-maternal interface around signalling of MRP in the horse (day 13) by means of mass spectrometry. A distinct influence of pregnancy was established, with 119 proteins differentially expres...
Proudman CJ, Trees AJ.Whole worm extract (WWE) and excretory/secretory (E/S) antigens of Anoplocephala perfoliata were characterised by SDS-PAGE and their use in the serodiagnosis of equine cestodosis was evaluated. An enzyme-linked immunosorbent assay (ELISA) was used to compare WWE and E/S antigen as the capture layer in an antibody capture ELISA. E/S antigen gave the best differentiation between sera from tapeworm-positive and tapeworm-negative horses. The E/S-ELISA was optimised and validated against sera from horses of known tapeworm status. This assay gave a diagnostic sensitivity of 68% (n = 38) and a specif...
Okabe T, Miyajima T, Nakagawa K, Tsukamoto S, Fujiwara K, Ikeguchi M.β-lactoglobulin forms a non-native α-helix during an early stage of folding. To address the role of the non-native structure in the folding process, we designed several mutants of equine β-lactoglobulin with reduced helical propensity in the non-native helix region. One of them, A123T, showed a similar structure to that of the wild-type protein; its folding kinetics was investigated by stopped-flow circular dichroism (CD) and fluorescence. Although A123T showed a reduced burst-phase CD intensity, its folding rate was similar to that of the wild-type protein, which indicated that the formati...
Blue HB, Blue MG, Kenney RM, Merritt TL.Forty uterine fluid samples were obtained from 4 mares classified as resistant to uterine bacterial infection. The uterus of each mare was flushed with 50 ml of saline solution during estrus and diestrus of successive estrous cycles. Bacteria or fungi were isolated from 4 samples, and 7 additional samples were obtained from a mare with active intrauterine infection. Fluid volumes obtained during estrus (means = 40.3 +/- 11 ml) tended to be greater than those recovered during diestrus (means = 36.8 +/- 7.9 ml), but the difference was not significant. Concentrations and yields of protein in reco...
