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1. 1. Electrophoretic studies are made of mature phase milk “whey” proteins and blood serum proteins of echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus). The echidna milk bands are designated AM, those of platypus AG. Some of the proteins are isolated and characterized. 2. 2. Echidna band A protein has some similarity to high cystine “whey” proteins. Band E protein (apparent M r 21,000) may be a β-lactoglobulin-like protein. Band M is lysozyme. Band C is serum albumin. Bands GK are transferrins. 3. 3. Platypus milk bands A, C, D, F and G are isolated. Bands F and G are transferrins. 4. 4. Lactose synthase and lytic activities are examined.

High-performance liquid chromatography has been applied to determination of modifier activity in alpha-lactalbumin (alpha-LA). An amino-bonded column separates uridine diphosphate (UDP) (product), UDPgalactose (substrate), and uridine monophosphate (UMP). From an aliquot of the same sample, a column for carbohydrate analysis separates lactose (the other product) and galactose-1,2-cyclic phosphate (Gal-c-P). Nucleotide peaks are detected by measurement of A262 and those of carbohydrate by 3H counting, the isotope originating from UDP-galactose-3H. A pH of 6.3 was taken as optimal for production of UDP since, at this level, the unwanted side reaction is minimized, by which UMP and Gal-c-P are formed. Thus, the conservation of substrate so effected may have contributed to an enhanced production of UDP. The reaction by which UDP and lactose are produced was linear for 120 min, as followed by UDP formation, but it continued to at least 300 min. Production of lactose was equivalent to that of UDP, when alpha-LA was the modifying protein. From a survey of seven other proteins, only lysozyme and ovalbumin showed ability to produce UDP. However, failure of the last two proteins to produce lactose indicates absence of modifier activity and demonstrates the need for monitoring both products.

1. 1. Problems in the isolation of whole casein from bovine milk are considered. A summary is given of our experiences in its isolation. 2. 2. The physical characteristics, sedimentation velocity, heterogeneity, absorptivity, nitrogen, phosphorus and carbohydrate contents of whole casein prepared from normal and sub-clinical mastitic milk samples by a variety of methods are compared. The methods are acid precipitation, high-speed centrifugation, with and without added calcium (II), and ammonium sulphate precipitation. 3. 3. A description is given of the low temperature ammonium sulphate procedure preferred for the isolation of whole casein, especially when it is to be used for subsequent fractionation for conformation and micelle studies. 4. 4. The question of the use of bovine casein as a paradigm for the caseins of other mammalian species and the need for further studies of the physical, chemical and biological properties of the caseins are discussed.

1. 1. Individual blood samples from 144 Bali (Banteng) cattle [Bos (Bibos) javanicus] in the Northern Territory of Australia and from 61 Bali cross cattle, were examined by zone electrophoresis to determine the variants of haemoglobin, serum albumin and transferrin that are present. 2. 2. Of the common cattle haemoglobin variants (A and B) only variant B occurs in the Bali cattle samples. A second variant, designated CBali, occurs in Bali cattle either as the heterozygote (B CBali) or as the homozygote, the frequencies of occurrence indicating a two-allele system of inheritance without dominance. The CBali cross samples may exhibit the homozygous or heterozygous A variant. 3. 3. The CBali variant has an electrophoretic mobility intermediate between those of the A and B variants at pH 8.6 and 9.1 but closer to B than to A (B > C > A). It appears to be similar in mobility to the C variants found in Indian Khillan (CKhillan) by Naik, Sukumaran and Sanghvi (Anim. Prodn. 1965 1, 275–277), and in Asian cattle by Oishi, Abe and Namikana (Immunogenet. Lett. 1968, 5, 170–173) and Abe, Mogi, Oishi, Tanaka and Suzuki (Proc. XIIth Europ. Conf. Anim. Blood Groups Biochem. Polymorphisms 1972, pp. 225–228), but appreciably different from those in Kenyan and Rhodesian cattle (CRhodesia) found by Braend (Anim. Blood Grps Biochem. Genet. 1971, 2, 15–21) and Carr (Rhod. J. agric. Res., 1964 3, 62–62A), respectively. It is also different in mobility from the C variant found by Winter, Mayr, Schleger, Dworak, Krutzler and Burger (Res. vet. Sci. 1984, 36, 276–283) in the mithun. 4. 4. On the basis of amino acid analysis and partial sequencing studies, it is concluded that the β-chain of the CBali variant differs from that of the A variant at position 18 (C His, A Lys) and from the B variant at positions 15 (C Gly, B Ser) and 119 (C Lys, B Asn). It differs in the α-chain from both A and B variants at positions 8 (C Ser; A, B Gly), 12 (C Thr; A, B Ala), 15 (C Asp; A, B Gly), and 71 (C Gly; A, B Glu). It is not identical with CRhodesia, nor with Yak I or II. 5. 5. A new serum albumin (designated DBali) moving more slowly than the C variant of Brahman cattle (Bell, personal communication, 1980) is observed in Bali blood samples. The only transferrin type present is D2. 6. 6. The results are discussed in the context of comparisons with other members of the Bovinae.

1. 1. Both α-lactalbumin and β-lactoglobulin-like proteins were detected in the whey fraction of feline milk by immunoblotting with rabbit antisera to α-lactalbumin and β-lactoglobulin, respectively. 2. 2. α-Lactalbumin was found to occur in both glycosylated and unglycosylated forms in approximately equal concentrations. No polymorphism of feline α-lactalbumin was found. 3. 3. Feline β-lactoglobulin-like proteins produced complex electrophoretic pattenrs that appears to be determined by three distinct loci. Between two and five genetic variants are expressed by each locus. 4. 4. Lysozyme was detected at levels of approximately 1 mg/ml in skim milk. 5. 5. The identification of the proteins as α-lactalbumin, β-lactoglobulin and lysozyme were confirmed by determination of N- terminal amino acid sequences.

1. A comparison is made of gel electrophoretic patterns of the "whey" proteins of the milk of red (Macropus rufus) and eastern grey (Macropus giganteus) kangaroos at various stages of lactation. Qualitative and quantitative changes occur with time during the mature phase of lactation of both types. Their onset is related solely to the stage of lactation. "Whey" proteins are isolated and characterised and the nature of protein changes determined for the first time. 2. The anodic electrophoretic pattern is divided into 6 main zones (designated A F in order of decreasing mobility) and 2 cathodic zones (G and H) that are only detected in the milk of M. giganteus. 3. Zones A, B and C are milk specific. Zone B is present throughout lactation in both species and is an alpha-lactalbumin. Zones A and C are present only in late lactation, zone C, usually, but not always, appearing first. Zone A is an alpha-lactalbumin in M. giganteus, but is not an alpha-lactalbumin in M. rufus. Zone C appears to be the same protein in both species and is possibly a beta-lactoglobulin. 4. Zone D is kangaroo serum albumin and zone E is possibly a beta 2-microglobulin. Zone F contains three main iron (III) binding bands whose relative intensity varies with stage of lactation. Their intensity differs from the corresponding blood serum transferrin bands. 5. Zone H of Macropus giganteus is a lysozyme. 6. Lactose is present in the milk, but is not the principal sugar. 7. The significance of the results is discussed.

1. 1. Zone electrophoretic patterns of milk samples have been examined from 713 mares, and classified in terms of 5 anodic zones and 1 cathodic zone at pH 8.5 (NaOHH 3 BO 3 ). 2. 2. Zone 1 protein exhibits genetic polymorphism, there being 5 variants, designated A–E. Zone 2 protein is the dominant whey protein and is designated a β-lactoglobulin. Zone 3 protein is equine serum albumin. Zone 4 contains equine α-lactalbumin and iron binding protein (probably transferrin). Zone 5 contains iron binding protein, probably lactoferrin. The cathodic zone 6 contains equine lysozyme. 3. 3. Methods of isolation and properties of these proteins are described and discussed. In particular, the amino acid sequence of the first 40 residues of equine β-lactoglobulin is compared with the corresponding sequences of the porcine and bovine proteins. The reason for the failure of peptide mapping to reveal the homology of the equine protein with other β-lactoglobulins is discussed.

SummarySkim milk samples from 126 Friesian and 147 Jersey cows in eight commercial herds were preheated at 85 °C for 30 min and concentrated to 200 g l−1total solids. A heat coagulation time–pH curve was determined at 120 °C for each treated sample. Heat coagulation times ranged from 1 to 50 min at the non-adjusted pH and 1 to 60 min at the pH of maximum stability. The following statistically significant effects were found. Maximum heat stability was affected by genetic variants of κ-casein (B > AB > A;P< 0·001) and β-lactoglobulin (B, AB>A;P< 0·05) whereas natural heat stability was affected only by κ-casein genetic variants (B > AB > A;P< 0·001). Maximum and natural heat stability were corre-lated positively with β-casein and κ-casein concentrations and were negatively correlated with αs1-casein and β-lactoglobulin concentrations. Milk from Jersey cows had greater maximum and natural heat stability than milk from Friesian cows. Differences were found between herds within breed for natural heat stability, but not for maximum heat stability. Maximum heat stability declined with age of the cow. The heat stability of skim milk samples taken from 40 Jersey cows in one of the herds was determined at 140 °C. A considerable variation was found in the coagulation time–pH curves. There was a difference in natural heat stability between κ-casein variants (B > AB;P< 0°05). Natural and maximum heat stability were correlated positively with urea concentration. No relationship was found between the heat stability of preheated concentrated skim milk and the heat stability of the original skim milk. The pH of skim milk samples was associated with αs1-casein genetic variant, age of cow, stage of lactation and concentration of γ-casein.

A new method, involving only gentle procedures, is described for the isolation of bovine β-casein. The optical rotatory dispersion (o.r.d.) and circular dichroism (c.d.) of the A1 variant, so isolated, are determined in the temperature range 2–60°C, at pH 6.9 (25°C) and I = 0.012 mol l −1 . Conformational analyses are made of the o.r.d. and c.d. results using the reference c.d. spectra of Brahms and Brahms, the Kronig—Kramers transform, and also of the c.d. results by the method of Provencher and Glockner. The conformations obtained are compared with those predicted for the amino acid sequence by the methods of Chou and Fasman and of Lim. It is concluded that β-casein contains 9 ± 2% α-helical structure and 25 ± 6% β-sheet at 2°C. A structural interpretation is proposed for the effect of increase of temperature on the o.r.d. and c.d., involving an increase in the proportion of β-sheet at the expense of aperiodic structure.

SummaryEffects of genetic variants of the milk proteins, αs1-casein, β-casein, κ-casein and β-lactoglobulin (βlg), on milk yield and composition, particularly the protein composition, were investigated in milk samples from 289 Jersey and 249 Friesian cows in eight commercial herds.Milk protein genotypes had no significant effect on yields over a complete lactation of milk and fat, but significant differences in fat content were detected for β-casein (B, A1B, A2 > A1A2) and βlg (B, AB > A) variants. Significant differences between β-lg variants were also found with total solids (B, AB > A), casein (B, AB > A), whey protein (A > AB > B) and βlg (A > AB, AC > B > BC) concentrations. Casein genotypes were not significantly different in total protein and casein concentrations but many differences were found in casein composition. αs1-Casein variants significantly affected αs1-casein (BC > B) and κ-casein (B > BC) concentrations. β-Casein variants affected concentration and proportion of β-casein (A1B, A2B > A1, A1A2, A2, B), αs1-casein (A1, A2 > B) and κ-casein (B > A2) and concentration of whey protein (A1 > most other β-casein variants). κ-Casein variants affected concentration and proportion of κ-casein (B > AB > A), proportion of αs1-casein (A > AB > B) and concentration of βlg (A > AB, B) and α-lactalbumin (A, AB > B). Differences in milk composition were found between breeds, herds and ages, and with stage of lactation. The potential use of milk protein genotypes as an aid in dairy cattle breeding is discussed.

A comparative study is made of the effect of pH on the urea denaturation of bovine serum albumin and ovalbumin. The denaturation process is followed by optical rotation, viscosity, gelation, sedimentation and diffusion measurements. In the pH range 3–10, bovine serum albumin shows a very rapid initial increase in laevorotation and viscosity in concentrated urea solution at 25°. Ovalbumin resembles bovine serum albumin in its behaviour in urea at low pH. Near neutrality and at alkaline pH values, ovalbumin undergoes a much slower change in these properties. The kinetics of the optical rotation change for ovalbumin generally resemble those reported by Simpson and Kauzmann , but there are some differences. Evidence is presented which indicates that these proteins behave in urea solution as random-coil polymers. The “unfolded” proteins show a tendency to aggregate slowly in 7 M urea. This is much more pronounced in neutral and alkaline solution and ovalbumin aggregates more readily than bovine serum albumin. The aggregation of “unfolded” bovine serum albumin appears to be due largely to a S-S-SH exchange reaction. This is only partly the case for ovalbumin. The effect of p -chloromercuribenzoate on S-S bonds is considered. The value of the techniques used is assessed. The importance of studies of the effect of pH in the classification of proteins with respect to thermodynamic and kinetic stability and in the elucidation of the mechanism of denaturation is stressed.

The effect of acid and urea on bovine serum albumin and ovalbumin has been investigated by ultraviolet spectrophotometry. Acid and urea bring about very similar shifts in the ultraviolet spectrum of bovine serum albumin. These changes are very rapid. The difference spectrum of bovine serum albumin at 287 mμ was investigated as a function of pH over the range 0.5–4.2. The effect of pH on ΔA287 was found to be closely similar to the effect of pH in this region on the sedimentation coefficient, viscosity and optical rotation. The effect of acid on the ultraviolet spectrum of ovalbumin is variable. The spectrum of ovalbumin in urea solution changes immediately at low pH. In urea solution at higher pH values the spectrum of ovalbumin changes with time. The change in A287 follows apparent first-order kinetics at both 25° and 30° at pH 6.1 and 7.8. The apparent order of the reaction with respect to urea at pH 7.8 and 30° is 7.8 ± 0.2, and at pH 6.1 and 25°, 7.6 ± 0.5. The change with time in the optical rotation of ovalbumin in 7 M urea at pH 6 and 25° is not simple first order. The half-time is inversely proportional to the 13.5 power of the urea concentration. These results are discussed in relation to current concepts of the mechanism of denaturation of bovine serum albumin and ovalbumin.

A study is made of the particle dimensions and homogeneity of ovine submaxillary gland mucoprotein by viscosity, sedimentation velocity and Archibald measurements. The substance exhibits hypersharp sedimentation velocity patterns and is polydisperse; s°20,ω is 8.5 ± 0.8 S and [η] is 3.4 ± 0.1 dl/g; ν is 0.685 ± 0.01 ml/g. Treatment of the data by several methods indicates an average molecular weight for the preparations studied of (1.0 ± 0.2) · 106. The dimensions J, the ellipticity, and V′, the effective hydrodynamic volume, of the equivalent ellipsoid are calculated by the method of Ogston to be 8.7 and 30 ml/g, respectively. It is concluded that the mucoprotein is probably a random coil with some rigidity. X-ray data seem to support this conclusion. Problems in the application of the Archibald method to molecules like ovine submaxillary gland mucoprotein are discussed.

Existing methods for the preparation of α-lactalbumin and β-lactoglobulin are examined critically. It is proposed that the most satisfactory method for the preparation of the whey protein fraction from bovine milk is (NH 4 ) 2 SO 4 fractionation. A number of new gentle procedures for the fractionation of the whey proteins are compared with some existing methods. The distribution of total protein at various stages is determined by total Kjeldahl nitrogen and the kind of protein by starch-gel electrophoresis. The effect of variables, such as pH, salt concentration and temperature on the separation of α-lactalbumin and serum albumin from β-lactoglobulin is studied. Differences in the behavior ( e.g. solubility) of genetic variants of β-lactoglobulin are noted. The solubility of each of the major whey proteins, under similar conditions to those used in the fractionation, is compared with its solubility in the whey protein mixture. The understanding gained of factors important in the fractionation is used in recommending new procedures for α-lactalbumin and β-lactoglobulin isolation.

The amino acid composition of the following variants of β-lactoglobulin has been determined: bovine A, B and C; ovine A and B; and caprid. Some of the amino acid differences within species have been characterized at the peptide level. The bovine A:Asp/B:Gly difference peptides have been located and studied. The peptides involving the assumed A:Val/B:Ala difference have not been detected. These observations are in essential agreement with those of Townend 14. Bovine B:Gln/C:His difference peptides have been isolated from the ‘tryptic core’. The extra glutamic acid residue of B compared with C, occurs in the amide form, glutamine, in the protein. The ovine B:His difference has been related to Gln in ovine A. It is therefore assumed that there is an A:Tyr/B:Glx substitution.

An improved method for the isolation of κ-casein, the rennin sensitive material of whole casein, is described. Acid casein is used as starting material and a comparatively high yield of product is obtained. The κ-casein has been characterized by sedimentation velocity, sensitivity to calcium and rennin, and by zone electrophoresis in concentrated urea.

1. 1. Each of two genetic variants of equine transferrin, D and R, is isolated from the blood of the heterozygote by a gentle fractionation procedure at pH 7.2. It is shown by step gradient polyacrylamide gel electrophoresis at pH 7.9 that each of these phenotypes exhibits two major bands (designated F, fast, and S, slow) and several minor bands. 2. 2. Components corresponding to these bands are separated by ion-exchange chromatography at pH 6.6 and 6.9 respectively for the D and R variants. 3. 3. The F and S components of each variant contain respectively four and two sialic acid residues. The nature of their heterogeneity is, at least in part, due to their varying sialic acid contents. It has not been possible to desialylate them completely by neuraminidase. 4. 4. On the basis of comparative studies of the tryptic and chymotryptic peptide maps of transferrins D and R it is concluded that there are at least two amino acid substitutions—D:R:Asp:Gly and Glu:Gly. 5. 5. These two substitutions are qualitatively in accordance with the difference in the electrophoretic mobility between the two variants at alkaline pH.

The presence of two new alpha-lactalbumins has been demonstrated in the colostrum of a single mare (Equus caballus, Persian Arab). They have been designated equine alpha-lactalbumin B and C, and that isolated previously from the milk of Australian horses (English Thoroughbred) as alpha-lactalbumin A. The primary structures of B/C have been determined by automatic Edman degradation of enzymatic cleavage of the oxidized protein. Cyanogen bromide cleavage of S-carbamoyl-methylated protein provided necessary overlapping peptides. Comparison of the sequences of B and C with that of A indicates 3 and 4 amino-acid exchanges, respectively. The phylogenetic difference of equine alpha-lactalbumin B/C from bovine alpha-lactalbumin B is indicated by 39 and 40 amino-acid exchanges, respectively. The structure-function relationship, calcium binding sites and variants of alpha-lactalbumin are discussed.

A method is described for the determination of lysozyme (muramidase) activity, whereby sensitivity is maximized by incubation of the reaction mixture (sample, buffer, and substrate (Micrococcus luteus] over an extended period. This approach is made feasible by exploiting our observation that the lytic reaction follows simple kinetic order during this time (e.g., 700 min for bovine lysozyme and 960 min for the eggwhite enzyme at low concentrations). After this period, the reaction rates diminish, indicating biphasic behavior, and eventually become negligible. The kinetic order may vary with both the type of lysozyme and the buffer system used. The limit of detection for bovine milk lysozyme is 100 pg/ml reaction mixture, equivalent to 6 ng/ml milk, for a 50-microliters sample (with reference to hen eggwhite lysozyme). With these limits, the method has proven valuable in our comparative studies, particularly for low levels of activity in bovine milk, but also in secretions and tissue extracts from various other eutherian, metatherian, and prototherian mammals. The method may also be applied to investigation of structure and function in modified forms of the enzyme.

Bovine α-lactalbumin B consists of one major and three minor components. They have been designated in terms of their decreasing order of mobility in starch gel electrophoresis at pH 7.7: α-lactalbumin B fast component (Component F), α-lactalbumin B major component (Component M), α-lactalbumin B slow component 1 (Component S1) and α-lactalbumin B slow component 2 (Component S2). Component F is eluted with Component M in chromatography on Sephadex G-75 at pH 6.3; whereas Components S1 and S2 are eluted together, but ahead of Component M. The order of elution from DEAE-Sephadex at pH 6.3–7.9 is Component S2, S1, M and F. These properties are exploited to separate the components. It is demonstrated that the minor components are true components, not resulting from interaction effects or conformational change. All are active in the lactose synthetase system, and have the same amino acid composition as the major component. Component F possibly differs from Component M in having one less amide group. Components S1 and S2 have carbohydrate moieties. The number of residues in Component S1 are 6–7 hexosamine, 6 hexose, 1 6-deoxyhexose, 1 sialic acid. Component S2 has the same number, except it has no sialic acid. The A variant has analogous minor components. Thus the AB heterozygote consists of 2 major and 6 minor components.

Methods are given for the isolation and crystallization of bovine β-lactoglobulin C. Properties of this new variant are compared with those of the A and B variants. There are differences in crystallization behaviour. The nitrogen contents and monomer molecular weights of the three variants are similar. The C variant shows little or no tendency to associate at pH 4.7 and low temperature, resembling the B variant and not the A variant. The laevorotation of C at pH 5.2 is slightly greater than that of the A or B variant but ao and bo are similar for all three variants. The ultraviolet absorption spectrum of C differs slightly from that of A or B. Optical rotatory dispersion and sedimentation studies indicate different stabilities for A, B and C and different tendencies for them to dissociate. The order of stability and dissociation are pH dependent. The three variants exhibit similar immunological behaviour.

Extensive optical rotation, viscosity, sedimentation, and spectroscopic studies of the urea denaturation of conalbumin and its iron complex are presented and considered in relation to the structure of these molecules. At low pH ( 5 M) solutions of conalbumin are very rapid, those of urea solutions of iron-conalbumin are much slower. In the pH range 8.8–10. iron-conalbumin is also considerably more stable towards urea than conalbumin. Experiments with cysteine present indicate that disulphide bonds are of importance in stabilizing the native configuration of both conalbumin and iron-conalbumin. Both acid and urea bring about similar rapid changes in the region of tyrosine and tryptophan absorption of the ultraviolet spectrum of conalbumin. The analogous spectral changes in urea solutions of iron-conalbumin are much slower. The change of absorbancy of iron-conalbumin at 292 mμ in concentrated urea solutions is first order with respect to time. The apparent order of reaction with respect to urea at pH 5.9 and 30° is 14. The spectroscopic changes accompanying urea denaturation of iron-conalbumin in the pH range 9.6–10.2 are complex and irreversible. Evidence is presented to show that at near-neutral pH, urea denaturation of conalbumin modifies, but does not destroy, the iron complex. Some preliminary experiments on the “renaturation” of conalbumin are reported.

A method for the modification of carboxyl groups in proteins is described. The water-soluble carbodiimide, 1-cyclohexyl-3(2-morpholinyl-(4)-ethyl) carbodiimide metho- p -toluene sulphonate (CMC), is employed. The procedure is tested on bovine serum albumin as a model. It is applied to demonstrate the probable role of carboxyl groups in the dimer-octamer association of bovine β-lactoglobulin A.

It is shown that either or both of two α-lactalbumins (designated A and B) may occur in the milk of Droughtmaster cattle. Their occurrence is genetically determined. The B variant has the same amino acid composition as the sole α-lactalbumin occurring in the milk of European breeds and the A variant differs from the B variant having one less arginine residue (0 vs. 1) and one more glutamine residue. There are two peptides in the map of the B variant that are not present in the map of A, and one peptide in B not present in A. These difference peptides have been isolated and their amino acid composition and electrophoretic mobility determined. One peptide has been subjected to Edman degradation. The data are considered in the light of the known partial sequence of α-lactalbumin of European breeds. It is concluded tentatively that the substitution of glutamine in A for arginine in B occurs at residue No. 10.

A comparative study is made of the sedimentation and optical rotatory dispersion behaviour of genetic variants of bovine β-lactoglobulin in sodium acetateacetic acid buffers near pH 5. Sedimentation velocity patterns of the A variant at pH 4.7 and low temperature are in agreement with the observations of others that the dimer unit of molecular weight 36 000 polymerizes rapidly to form predominantly an octamer (for a review see ref. 1). The dimer of the B variant shows only a small tendency to undergo polymerization. The relationship of sedimentation coefficient to protein concentration is employed as a useful index of polymerization in several cases wherein association is sufficiently weak that it is not readily apparent from the sedimentation patterns. The weight average molecular weight of the A and B variants is measured at pH 4.7 in the range 4–32° using the approach to sedimentation equilibrium principle of A rchibald . A method is presented for treating the data to determine the equilibrium constant of the dimer-octamer reaction in the A variant. Values of relevant thermodynamic functions are determined. Sedimentation equilibrium measurements are made at low concentration for the A, B and C variants at pH 5.4 and 20°. There is some evidence of dissociation of the dimer to the monomer under these conditions contrary to the conclusions of others. The effect of the dimer-octamer reaction on the optical rotatory dispersion properties of the A variant is studied extensively. The reaction is accompanied by a considerable change in [α]λ and ao, but only a small change in bo. It is shown how the former two changes may be used to calculate the equilibrium constant and thermodynamic functions of the reaction. The results obtained are in good agreement with those obtained by other weight average methods. The method is a valuable one and applicable generally in favourable cases of associating-dissociating systems. Possible conformational changes in the dimer of the A variant to facilitate octamerization are discussed.

THE most satisfactory measure of the ‘character’ of a bond in an aromatic compound is probably given by its ‘bond-order’1. Although this is a theoretical quantity, its significance is demonstrated by its relationship to bond-length1 and to other bond properties which can be measured experimentally. It has also been shown2,3 that there is a smooth-curve relationship between bond-orders and the ‘corrected’4 redox potentials of the corresponding o-quinones. As redox potentials can be measured fairly readily, this relationship offers a method whereby certain bond-orders can be evaluated experimentally.

An electrophoretic examination is made of milk samples taken from eight Bali (banteng) cattle, Bos (Bibos) javanicus, at Beatrice Hills, Northern Territory, Australia. Starch-gel electrophoresis at pH 8 · 5 (NaOH-HB0 buffer) and filter-paper electrophoresis at pH 8· 6 (diethylbarbiturate buffer) indicate that all samples contain a new α-lactalbumin variant, designated a-lactalbumin C. The order of mobility for bovine variants is A > B > C. The C variant differs from the common B variant in having one more amide residue (substitution of Asn for Asp or GIn for Glu). © ASEG. Examination of milk samples by urea-starch-gel electrophoresis at alkaline pH indicates that there is a new asrcasein variant, designated α-casein E;, present in some samples. No new K-casein variant is detected by this method (all samples typing as K-casein B). A new variant of β-casein, designated Ais detected by urea-starch-gel electrophoresis at low pH. The variants of milk proteins observed in this paper and in Bell et al. (1981) are discussed in relation to those of other members of the Bovinae, especially the yak, Bos (Poephagus) grunniens.

SINCE 1902, when Ramsden1 described the effects of urea on “proteids”, there has been much speculation regarding the molecular weights of proteins in urea solution. Some workers have claimed that aggregation does not take place in the urea denaturation of ovalbumin and bovine serum albumin. In spite of this, it is well known that their urea solutions frequently form gels when the protein concentration is high. Recently, Kauzmann et al. 2,3 have studied the viscosity changes accompanying the urea denaturation of these proteins and the gelling time for ovalbumin. Under certain conditions an aggregation dependent upon concentration occurred, and this was interpreted in terms of hydrogen bonding and inter-molecular disulphide bonds (the latter through the —SS— and —SH exchange reaction of Huggins, Tapley and Jensen4).