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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.

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.

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.

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.

A method for the prreparation of myosin from carp red muscle and from mullet white and red muscles is described. The mullet myosin obtained is only very paritally centrifugated off at ionic strenght 0.05 and pH 7.2 at 25, 000 × g itis necessary to lower the pH to 6.4 in order to complete thye precipitation. Apart from this anomaly, the preparations isolated from carp and mullet muscles behave as rabbit myosin from the viscosimetric, electrophoreic and ultracentrifugal points of view. The rate of sedimentation at zero concentration is 6.03 ± 0.12 S, somewhat lower than the corresponding value for cod myosin and about identical with recent values found for rabbit myosin. Electrophoresis of fish myosin in borate buffer at pH 10.5 gives much more symmetrical gradients than in phosphoate buffer of pH 7.2 suggesting that the former solution could be more favourable for the study of the monomeric form for myosin. The stability of the myofibrils of white and red fish muscles in presence of extractants of high ionic strength appeared to be very different. Their distruption is easy in the case of white muscle while the behaviour of red muscle is reminiscent of rabbit muscle. The submicroscopic organisation of the myofibrils in white and red fish muscles is obviously very different.

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.

It is shown by starch-gel electrophoresis of samples of skim milk from individual ewes that the major part of the whey proteins migrates in three regions at pH 7.6 (phosphate buffer, I 0.2). In the region of highest mobility there is only one band and it represents serum albumin. The second region contains only one band and it corresponds to α-lactalbumin. In the third region there may be one or two bands, each of which is a genetic variant of β-lactoglobulin. These conclusions are confirmed by electrophoretic and immunological studies of total whey protein solutions and fractions isolated therefrom. The faster-moving β-lactoglobulin at pH 7.6 is designated ovine β-lactoglobulin A and the slower one ovine β-lactoglobulin B. Their isolation and properties are described. The A variant can be crystallized readily at pH 5.2. Attempts to crystallize the B variant have failed. Neither variant associates at pH 4.6 and low temperature, thus resembling the bovine B and C variants rather than the bovine A variant. There are marked differences in the ultraviolet spectra due to the difference in tyrosine content. The optical rotatory properties, molecular size and immunological properties are similar to those of the bovine species.

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.