SAP-6-Val proteins and methods

A novel hydrophobic surfactant-associated protein mixture, i.e., a SAP-6 proteins, has been isolated from pulmonary animal tissue. A small, novel pulmonary hydrophobic surfactant-associated SAP-6-Val protein having a molecular weight of about 6,000 daltons as determined by SDS-PAGE and about 3,500-4,000 daltons as determined by tricine-SDS-PAGE has been further isolated from the SAP-6 protein mixture. The amino acid residue compositions of the SAP-6-Val protein for human and bovine have been determined and disclosed. When a SAP-6-Val protein is combined with phospholipids, it enhances the surfactant-like activity of the phospholipids in lungs of animals and, therefore, uniquely imparts to the mixture significant pulmonary biophysical activity. Such a mixture results in enhanced adsorption of the phospholipids with properties similar to that of natural pulmonary surfactant material. SAP-6-Val proteins in combination with phospholipids is highly useful for replacing or supplementing natural pulmonary surfactant material for reducing or maintaining normal surface tension in lungs, and especially in lungs of patients suffering from hyaline membrane disease, HMD, or other syndromes associated with the lack or insufficient amounts of natural pulmonary surfactant material. A mixture of a SAP-6-Val protein and phospholipids may be administered as an aerosol spray or in aqueous normal saline with or without calcium chloride for treating or preventing HMD and other surfactant deficiency states. Also disclosed are methods of isolating the noncanine SAP-6-Val proteins from the SAP-6 proteins isolated from animal tissue.

FIELD OF THE INVENTION 
The present invention relates to isolated SAP-6-Val proteins, methods of 
isolating and using same and medicaments formed therewith. 
BACKGROUND 
Hyaline membrane disease, HMD, is a common disorder of premature infants 
and is related to diffuse atelectesis, hypoxia and resultant respiratory 
impairment. More particularly, HMD relates to the lack of vital pulmonary 
material necessary for reducing surface tension in the airways of the 
alveoli. As a result, the alveoli or terminal respiratory sacs of patients 
suffering from HMD normally collapse. And, because the surface tension at 
the gas-liquid interface in HMD patients is elevated, their alveoli or 
terminal respiratory sacs are very difficult to reinflate. Consequently, 
HMD may be associated with significant morbidity and mortality, especially 
in premature infants. 
Present treatments of HMD focus on using high concentrations of oxygen, 
positive pressure and/or mechanical ventilation to maintain adequate 
oxygenation. These therapies are complicated by oxygen and pressure 
related injuries as well as injuries resulting from the need to 
mechanically access the airway via endotracheal tubes. More recent 
studies, however, have supported the use of replacement pulmonary 
surfactant material for therapy of HMD and other syndromes associated with 
the lack of pulmonary surfactant material. Such therapy has included 
heretofore the use of aerosolized or liquid synthetic phospholipid 
mixtures, natural pulmonary surfactant material and various preparations 
of surfactant material prepared from animal lung. Surface tension lowering 
ability of the naturally derived preparations is in general better than in 
the synthetic lipid preparations. Also, preliminary studies using modified 
bovine surfactants have been promising. Problems with human and animal 
pulmonary preparations, however, have occurred which include lack of 
variability, possible infection and immunologic risks. Obviously, when 
treating patients for any disease including HMD, it is imperative to 
include only those active substances necessary to minimize possible 
immunologic consequences of therapy to the patients. Unfortunately, 
because the natural pulmonary surfactant material and the preparations 
available heretofore are in crude form, they are less specific and 
associated with possibly greater immunologic risks. 
Natural pulmonary surfactant material is a complex material composed 
primarily of phospholipids and surfactant-associated proteins or 
apolipoproteins. The phospholipids, mainly phosphatidylcholine (PC), 
disaturated phosphatidylcholine (DSPC) and phosphatidylglycerol (PG), are 
of paramount importance for the physiological role of natural pulmonary 
surfactant material in reducing surface tension in the alveoli. 
Phospholipids, of which DSPC is the principal component, are synthesized 
in the endoplasmic reticulum of Type II epithelial cells, packaged into 
lamellar bodies, then secreted into the alveolar space by an exocytotic 
process. Several of the phospholipids are apparently not catabolized and 
resynthesized, but rather it is presently believed that they are 
reutilized primarily as intact molecules and constitute the major 
components of the naturally existing pulmonary surfactant material. 
With respect to the surfactant-associated proteins or apolipoproteins, 
there is considerable disagreement as to their identity and utility. 
Nonetheless, there is increasing agreement among those with medical 
expertise in this area that in addition to the lung surfactant 
phospholipids, at least some of these apolipoproteins are vital for the 
full biological activity of the natural pulmonary surfactant material in 
reducing surface tension in the alveoli. 
Surfactant-associated proteins or apolipoproteins include both serum and 
lung specific proteins. The major lung specific surfactant-associated 
protein of 30-40,000 daltons identified in lung surfactant by King et al, 
Isolation of Apoproteins from Canine Surfactant Material, Am J Physiol 
244:788-795, 1973, is a glycoprotein rich in glycine and containing 
collagen-like regions rich in hydroxyproline. This protein, herein called 
SAP-35, is synthesized from 28-30,000 dalton translation products which 
undergo glycosylation, hydroxylation of proline residues and 
sulfhydryl-dependent cross-linking to form large oligomers which can be 
detected in the airway. Proteolytic fragments of SAP-35 have been 
identified in protein preparations isolated from lavage of patients with 
alveolar proteinosis and from other mammalian surfactants migrating as 
proteins of small molecular weight by Whitsett et al, Characteristics of 
Human Surfactant-Associated Glycoprotein(s) A, Pediatr Res 19:501-508, 
1985. While the glycoprotein SAP-35 binds phospholipids and may confer the 
structural organization of tubular myelin to surfactant lipids, it remains 
unclear whether SAP-35 is required for the biophysical activity of 
surfactants. See King et al, Metabolism of the Apoproteins in Pulmonary 
Surfactant, J Appl Physiol 42:483-491, 1977. 
Smaller lung specific surfactant-associated proteins have also been 
identified from a variety of mammalian surfactants. King et al, Am J 
Physiol 223:715-726, 1972, previously described a 10,000-12,000 dalton 
protein in pulmonary surfactant material; however, the origin of this 
protein or its distinction from others was not clarified. This protein 
described by King et al is now believed to be a fragment of the major 
glycoprotein SAP-35. Smaller surfactant-associated proteins, other than 
that reported by King et al, have been identified in alveolar lavage 
material from a number of species and with molecular weights of 
approximately 10,000 daltons in dog and rabbit, 10,500-14,000 daltons in 
rat, 11,500-16,500 daltons in pig, and 10,000 daltons in cow. 
The nature and relationships among these various surfactant-associated 
proteins (SAPs) and the larger protein, SAP-35 or its fragments, have not 
been established. Nevertheless, the work of Suzuki et al, J Lipid Res 
23:53-61, 1982, suggested that a small 15,000 daltons protein in pig 
alveolar lavage had a greater affinity for lipid than SAP-35. Suzuki et al 
unfortunately did not distinguish the proteins from the SAP-35 or its 
fragments or demonstrate if there exists surfactant properties in a 
purified state. Rather, Suzuki et al only suggested that this 15,000 
dalton protein is possibly a physiological regulator for the clearance of 
alveolar phospholipid. Claypool et al, J Clin Invest 74:677-684, 1984, 
suggested that a small unidentified protein, isolated from rat alveolar 
lavage, increased the uptake of liposomes by cultured Type II epithelial 
cells. Work by Wang et al, Amino Acid Composition of Low Molecular Weight 
Hydrophobic Surfactant Apoproteins, Fed Proc 44:1024 (abstract), 1985, 
described two distinct small molecular weight proteins in surfactant from 
rat that are ethanol soluble. But like Suzuki et al, Wang et al failed to 
purify, identify activity, or characterize the protein to homogeneity. 
Wang et al instead suggested that these small molecular weight proteins 
may be involved with surfactant recycling. It also has been suggested that 
the smaller molecular weight proteins, such as those discussed above, 
possibly arise as proteolytic fragments of the larger SAP-35 molecule At 
present, however, it is unclear whether SAP-35, one or more of the smaller 
proteins, or all proteins together are active components imparting 
biophysical activity to natural mammalian pulmonary surfactant material. 
In view of the present state of the art, there obviously are needs to 
clarify the nature and role of the surfactant-associated lung specific 
proteins and to determine the most effective means for treating and 
preventing HMD and other syndromes associated with lack or insufficient 
amounts of pulmonary surfactant material to maximize HMD therapy and to 
eliminate the disadvantages associated with HMD therapy available 
heretofore. 
SUMMARY OF THE INVENTION 
In brief, the present invention alleviates and overcomes the above problems 
and shortcomings of the present state of the art through the discovery of 
a novel hydrophobic surfactant-associated proteins isolated from animal 
tissue, hereinafter "SAP-6 proteins". The novel SAP-6 proteins comprise 
two hydrophobic surfactant-associated proteins. It has been discovered 
that when the SAP-6 proteins are combined with phospholipids to form a 
novel preparation, such preparation has significant pulmonary biophysical 
surfactant activity that can be utilized to effectively treat and prevent 
HMD and other syndromes associated with the lack or insufficient amounts 
of natural pulmonary surfactant material. Although it is presently 
believed that the SAP-6 proteins are lung specific, their pulmonary 
biophysical surfactant activity is believed not to be species specific. 
The SAP-6 proteins therefore can be purified from animal tissue, 
specifically pulmonary tissue or amniotic fluid, extracted from a variety 
of animals, such as canine, bovine, human, porcine, rabbit, rat and the 
like. Notwithstanding, the concentrations of the SAP-6 proteins in 
pulmonary tissue and lavage are probably greater than that found in 
amniotic fluid. With respect to other animal tissues or fluid, however, 
the SAP-6 proteins are believed to be absent or present in substantially 
smaller or undetectable concentrations. 
In a further discovery, a novel SAP-6-Val protein has been isolated from a 
mixture of the SAP-6 proteins in substantially pure form, as described 
hereinafter. The novel SAP-6-Val protein is termed SAP-6-Val on the basis 
of an unique polyvaline domain. The novel SAP-6-Val protein comprises 
approximately 33-38 amino acid residues and includes within its protein 
structure a hydrophobic region rich in valine residues. The hydrophobic 
region of the SAP-6-Val protein is believed to play an important role in 
the association of the SAP-6-Val protein with membranes, and it is within 
this hydrophobic region that the unique polyvaline domain resides. The 
polyvaline domain originally contains a first stretch of about 6 adjacent 
valine residues and a second stretch of about 3 adjacent valine residues 
separated from the first stretch by about 2 hydrophobic amino acid 
residues. The hydrophobic region and stretches of adjoining valine 
residues are uniquely and inherently characteristic for the SAP-6-Val 
proteins and are believed to be common to different species, as depicted 
by the sequences identified for human, bovine and canine reported 
hereinafter. 
In a yet further discovery, it has been realized that when the SAP-6 
proteins are initially isolated in a mixture from animal tissue, it 
compromises the SAP-6-Val protein and a second hydrophobic SAP-6 dalton 
surfactant-associated protein, as well as multimers of both such proteins. 
Once the SAP-6-proteins have been isolated from animal tissue, the two 
SAP-6 proteins can be quite easily separated from the larger multimers 
thereof by, for instance, gel electrophoresis migration. And, based upon 
this gel separation technique, it is believed that the two SAP-6 proteins 
and in particular the SAP-6-Val protein constitute the predominant 
components of the mixture of isolated SAP-6 proteins. When utilizing gel 
electrophoresis migration in the absence of sulfhydryl reducing agents, 
however, increased amounts of the larger, hydrophobic protein multimers 
are detected. 
The simple molecular weights of the two SAP-6 proteins, i.e., the SAP-6-Val 
protein and the other hydrophobic SAP-6 protein of the SAP-6 mixture, are 
about 6,000 daltons for SAP-6-Val and about 6,000 daltons for the other 
SAP-6 protein as determined in a polyacrylamide gel containing sodium 
dodecyl sulfate (SDS-PAGE). The molecular weights for the larger, protein 
multimers are about 14,000, about 20,000 and about 26,000 daltons in 
SDS-PAGE. While the simple molecular weight for the SAP-6-Val protein is 
about 6,000 daltons in SDS-PAGE, it is about 3,500-4,000 daltons in 
tricine-SDS-PAGE. It should be readily apparent to those versed in the 
art, however, that since gel electrophoresis migration has been employed 
to determine the molecular weights for these novel proteins, the molecular 
weights are only estimates based upon relative migrations. 
The SAP-6 proteins and the larger, hydrophobic protein multimers thereof 
can be further characterized as having the ability to enhance the 
surfactant-like activity of phospholipids in lungs of animals, and as 
being substantially resistant to protease enzymes, endoglycosidase F, and 
collagenase. Furthermore, it is believed that when the SAP-6 proteins are 
combined with phospholipids, the SAP-6 proteins uniquely enhance the 
surfactant properties of the phospholipids thus imparting to the 
combination significant pulmonary biophysical surfactant activity. 
Likewise, when the SAP-6-Val protein is combined with phospholipids, the 
SAP-6-Val protein has the unique ability to enhance the surfactant-like 
activity of phospholipids. As a result of this remarkable property, such 
combinations are highly useful for replacing or supplementing natural 
pulmonary surfactant material for reducing or maintaining normal surface 
tension in the lungs, especially in the lungs of patients suffering from 
HMD and other syndromes associated with the lack or insufficient amounts 
of natural pulmonary surfactant material. And, since the SAP-6 proteins 
and especially the SAP-6-Val proteins of this invention can be highly 
purified from animal tissue, the immunologic risks currently associated 
with the less pure preparations available heretofore for treating or 
preventing HMD or related syndromes are substantially reduced. 
In accordance with another discovery of the present invention and as 
earlier reported herein, it is believed that there are at least two 
hydrophobic surfactant-associated proteins which form the SAP-6 mixture, 
and that such proteins have the unique ability to enhance the 
surfactant-like activity of phospholipids in lungs of animals. It is 
presently believed that these two hydrophobic proteins, i.e., the 
SAP-6-Val 6,000 dalton protein and the other SAP-6 6,000 dalton 
hydrophobic surfactant-associated protein, co-elute together, co-purify 
together via gel electrophoresis migration in SDS-PAGE, and have similar 
biophysical properties. Therefore, it is to be understood that the terms 
"surfactant-associated protein," and "SAP-6 protein(s)" are used 
interchangeably herein and that, whenever referenced herein, they are 
meant to include any small hydrophobic surfactant-associated protein that 
has the unique ability to enhance the surfactant-like activity of 
phospholipids in lungs of animals, that has a simple molecular weight of 
about 6,000 daltons determined by SDS-PAGE and which is substantially 
resistant to protease enzymes, endoglycosidase F and collagenase. 
The present invention further resides in a method of separating from animal 
tissue the novel SAP-6 proteins which involves separating the animal 
tissue into a particulate fraction and a liquid fraction, and extracting 
from the liquid fraction the novel SAP-6 proteins in a substantially pure 
state. The methods of this invention are further concerned with separating 
the SAP-6-Val protein from the mixture of SAP-6 proteins, for instance, 
via dialysis, and also separating the larger, novel hydrophobic multimers 
from the SAP-6 proteins. As already mentioned above, multimer separation 
can be accomplished by, for instance, gel electrophoresis migration or 
other suitable techniques. 
The present invention still further contemplates novel medicaments, 
preparations and methods employed to treat animals, including human 
infants, suffering from HMD and other syndromes related to the lack or 
insufficient amounts of natural pulmonary surfactant material. Also 
contemplated by the present invention are novel antibodies and antisera 
directed against the SAP-6 proteins and multimers thereof. 
Accordingly, it can be appreciated that the present invention provides a 
solution to the art that has long sought to understand natural pulmonary 
surfactant material and effective means to treat or prevent HMD and other 
syndromes associated with the lack or insufficient amounts of natural 
pulmonary surfactant material. 
The above features and advantages will be better understood with reference 
to the Figs., Detailed Description and Example set out hereinbelow. It 
will also be understood that the compositions and methods of this 
invention are exemplary only and are not to be regarded as limitations of 
this invention.

DETAILED DESCRIPTION OF THE INVENTION 
By way of illustrating and providing a more complete appreciation of the 
present invention and many of the attendant advantages thereof, the 
following detailed description is provided concerning the novel SAP-6 
proteins, the novel SAP-6-Val protein, the novel medicaments, the novel 
antibodies and antisera, and the novel methods of isolation and 
utilization thereof. 
The present invention provides novel, hydrophobic surfactant-associated 
SAP-6 proteins isolated from animal tissue, specifically from pulmonary 
tissue and amniotic fluid. The novel SAP-6 proteins consists essentially 
of two small, novel hydrophobic surfactant-associated proteins, i.e., the 
SAP-6-Val protein and a second hydrophobic SAP-6 protein, and larger, 
hydrophobic protein multimers of both such proteins. The SAP-6 proteins 
have simple molecular weights of about 6,000 daltons and the larger, 
hydrophobic protein multimers thereof have molecular weights of about 
14,000, about 20,000 and about 26,000 daltons as determined by SDS-PAGE. 
As earlier reported herein, one SAP-6 protein i.e., the SAP-6-Val protein, 
also has a simple molecular weight of about 3,500-4,000 daltons as 
determined by tricine-SDS-PAGE. It should be realized that other multimers 
may exist but in smaller undetectable concentrations. As used herein, 
"simple molecular weight" refers to the molecular mass of what is thought 
to be the smallest polypeptide chain after sulfhydryl reduction which 
serves as a repeating building block for a multimer. 
To determine the molecular weights and separate the SAP-6 proteins from the 
larger multimers, for instance, a gel formed with about 3-27% 
polyacrlyamide, in particular about 15% polyacrylamide (PAGE), and about 
2% of sodium dodecyl sulfate (SDS) can be used to separate and determine 
the molecular weights of the novel proteins in SDS-PAGE gel. See FIG. 1. 
For comparison, low molecular weight protein markers such as trypsin 
inhibitor (6,200), lysozyme (14,000), beta-lactalbumin (18,400), 
alpha-chymotrypsin (25,700) and ovalbumin (43,000) can be used. These can 
be obtained from BRL, Inc., of Bethesda, MD. Although the SAP-6 proteins 
are detected in relatively greater amounts than the multimers, an increase 
is observed in the larger protein multimer forms when the gel separation 
is performed on the SAP-6 proteins in the absence of sulfhydryl reducing 
agents, such as beta-mercaptoethanol, dithiothreitol (DTT), or after 
reduction and alkylation. Presently, it is believed sulfhydryl bonding, 
non-sulfhydryl aggregation and/or interpeptide bonding of a fraction of 
the SAP-6 proteins account for the larger, hydrophobic multimer forms. As 
earlier discussed in the summary section hereof, it should be understood 
that the molecular weights are only estimates based upon relative 
migrations and that other suitable techniques may be employed to ascertain 
the molecular weights. 
Throughout the specification, the SAP-6 proteins including the larger 
multimers thereof, collectively as "SAP-6 proteins," are characterized as 
being hydrophobic and surfactant-associated proteins. As used herein, 
"hydrophobic," refers to solubility in non-polar solvents, such as 3:1 
ether/ethanol, chloroform, chloroform/methanol in various ratios, such as 
3:1, and having an abundance of hydrophobic non-charged amino acids. The 
terms "SAP-6 protein," and "surfactant-associated protein," as used 
herein, refers to proteins associated with binding to or co-purifying with 
the phospholipid components of mammalian surfactants during centrifugation 
in isotonic solution. With respect to the term "multimer" as used herein 
in connection with the larger hydrophobic proteins, it refers to either 
covalent or non-covalent aggregates of 6,000 dalton SDS-PAGE SAP-6 
proteins which form oligomers of varying molecular weights, such as 
dimers, trimers, quatramers, etc. 
SAP-6 proteins can be further characterized as being substantially 
resistant to protease enzymes (trypsin, chymotrypsin and staph V-8), 
endoglycosidase F, and collagenase. It has been discovered that SAP-6 
proteins are not degraded nor are their sizes or size heterogeneities 
significantly altered by these enzymes. 
In further characterizing the SAP-6 proteins, they are found to have unique 
localization in lamellar bodies of pulmonary type II cells and surfactant 
in a variety of mammalian species as well as human amniotic fluid near 
term gestation. 
With respect to the determination of the amino acid composition of a SAP-6 
protein, reduced and alkylated purified samples are hydrolyzed in about 
300 microliters of constant boiling 5.7N HC1 containing 0.3% phenol and 
0.01% beta-mercaptoethanol at about 150.degree. C. under vacuum for about 
24 hours. The amino acids are determined using a Beckman 6300 amino acid 
analyzer with a SICA 7000A Integrator. 
Table I illustrates the amino acid compositions that have been determined 
for a bovine SAP-6-Val protein and a canine SAP-6-Val protein. It should 
be understood to those skilled in the art that since the amino acid 
compositions have been determined using a Beckman 6300 analyzer, the amino 
acid residue compositions per protein molecule are only estimates based 
upon this analytical technique. 
TABLE I 
______________________________________ 
Approximate Amino Acid Compositions of SAP-6-Val Proteins 
Amino Acid Residues 
Per Protein Molecule 
Amino Acid Residues 
Bovine Canine 
______________________________________ 
Cysteine (CM-Cys) 
about 3.6 about 1.6 
Aspartic acid (ASX) 
about 2.1 about 0.5 
Threonine (The) about 0.2 about 0.3 
Serine (Ser) about 0.6 about 4.0 
Glutamic acid or about 0.3 about 0.4 
Glutamine (Glx) 
Proline (Pro) about 4.3 about 4.6 
Glycine (Gly) about 4.1 about 5.1 
Alanine (Ala) about 2.3 about 2.3 
Valine (Val) about 23.1 about 19.2 
Methionine (Met) about 1.8 about 1.7 
Isoleucine (Ile) about 4.7 about 5.9 
Leucine (Leu) about 13.8 about 13.9 
Tyrosine (Tyr) about 0.2 -- 
Phenlyalanine (Phe) 
about 0.3 about 1.9 
Histidine (His) -- -- 
Lysine (Lys) about 2.0 about 2.0 
Tryptophan (Trp) -- -- 
Arginine (Arg) about 1.9 about 2.0 
Estimated Mol. Wt. 
6588 6441 
on SDS-PAGE of dimers 
of SAP-6-Val proteins 
______________________________________ 
CM-Cys = carboxymethyl cysteine. 
Two distinct SAP-6 proteins essentially form the SAP-6 mixture of the 
instant invention, i.e., a SAP-6-Val protein and a 6,000 dalton 
hydrophobic surfactant-associated protein. In accordance with this 
invention, the SAP-6-Val protein has been isolated from the SAP-6 mixture 
in virtually pure form and identified herein. The predicted sequence for a 
human SAP-6-Val protein and the partial NH.sub.2 -terminal end amino acid 
sequences of SAP-6-Val proteins isolated from human, bovine and canine 
tissue are also characterized hereinbelow. 
The SAP-6-Val protein is termed SAP-6-Val on the basis of an unique 
polyvaline domain. The SAP-6-Val protein is believed to be comprised of 
approximately 33-38 amino acids and is thought to either begin with the 
amino acid residue isoleucine (Ile), phenylalanine (Phe) or glycine (Gly) 
following the NH.sub.2 -terminal residue or include the amino acid residue 
isoleucine (Ile) within the first few residues following the NH.sub.2 
-terminal residue. Since the SAP-6-Val protein is derived from larger 
precursor proteins, proteolytic processing of the precursor proteins for 
SAP-6-Val can result in different amino acid residues located adjacent the 
NH.sub.2 -terminal residue. For example, leucine (Leu), phenylalanine 
(Phe) and glycine (Gly) have been detected in the first cleavage of 
SAP-6-Val proteins. Thus, it should be understood that SAP-6-Val may 
include isoleucine (Ile), phenylalanine (Phe), glycine (Gly), leucine 
(Leu), arginine (Arg), etc. as one of the first amino acid residues 
immediately following the NH.sub.2 -terminal residue. With respect to the 
amino acid residues located near the C-terminal end of a SAP-6-Val 
protein, their precise characterization remains unclear. Nevertheless, 
approximately 33-38 amino acid residues are thought to extend between the 
C-terminal and NH.sub.2 -terminal residues. 
A SAP-6-Val protein of the instant invention includes a hydrophilic region 
extending from the N-terminal residue and a hydrophobic region of 
approximately 25 amino acids extending between the Leu.sub.11 and 
Leu.sub.33 residues, as indicated by the sequence below. This hydrophobic 
region of about 25 amino acid residues is valine-rich, as suggested by the 
SAP-6-Val proteins tabulated in Table I and the sequences reported below, 
and is believed to play an important role in the association of the 
SAP-6-Val protein with membrane. The hydrophobic region contains a first 
stretch of about 6 adjacent valine amino acid residues and a second 
stretch of about 3 adjacent valine amino acid residues which are separated 
from one another by about 2 hydrophobic amino acid residues, such as 
leucine (Leu), isoleucine (Ile) and valine (Val). The hydrophobic region 
and stretches of adjoining Valine residues are uniquely and inherently 
characteristic for the SAP-6-Val protein and are believed to be common to 
different species, as depicted by the sequences identified for human, 
bovine and canine below. 
While the molecular weight of SAP-6-Val as determined by SDS-PAGE is about 
6,000 daltons, its molecular weight is about 3,500-4,000 daltons when 
determined by tricine-SDS-PAGE. This lower molecular weight for SAP-6-Val 
is also confirmed by the types and number of amino acid residues, i.e., 
about 33-38, in the composition of SAP-6-Val. 
The discrepancy in the electrophoretically determined molecular weights of 
SAP-6-Val is believed to be due in part to the superior resolution 
capability of the tricine-SDS-PAGE technique. The heterogeneity of the 
size of the SAP-6-Val protein in SDS-PAGE may result from the fact that 
SAP-6-Val may form aggregates or oligomers which migrate as larger forms 
as determined by SDS-PAGE. Regardless of which electrophoretic technique 
is selected to determine the molecular weight of SAP-6-Val, however, both 
molecular weights represent inherent identifying characteristics for 
SAP-6-Val as isolated in accordance with the methods of this invention. 
As already indicated above, the predicted amino acid sequence for SAP-6-Val 
protein is thought to begin at the Ile amino acid residue or at a few 
amino acid residues before the Ile amino acid residue and extend to about 
the Leu.sub.33 amino acid residue. The hydrophobic domain of amino acids 
including amino acid residues Leu.sub.11 to Leu.sub.33 is thought to be 
compatible with a membrane-associated region of approximately 25 amino 
acids. This hydrophobic region contains the characteristic stretches of 
repeating valine residues. A predicted amino acid sequence for a human 
SAP-6-Val protein is as follows: 
##STR1## 
Presently, the sequences for what is believed to be the first 18, the first 
25 and the first 23 amino acid residues following the amino-terminal 
residue for a SAP-6-Val human, bovine and canine protein, respectively, as 
ascertained from standard sequencing techniques, are as follows. 
For human, the first 18 amino acid residues after the amino-terminal 
residue are 
##STR2## 
Thus in view of the above amino acid sequence for the human SAP-6-Val 
protein, it should be appreciated that the human SAP-6-Val sequence may 
comprise 
##STR3## 
The Amino acid residues designated in paretheticals above and herein 
throughout represent optional or alternate or unconfirmed amino acid 
residues. 
For bovine, the first 25 amino acid residues after the amino-terminal 
residue are 
##STR4## 
For canine, the first 22 amino acid residues after the amino-terminal 
residue are 
##STR5## 
In the above sequences, the amino acid residues having alternately 
designated amino acid residues in parentheticals underneath are not yet 
confirmed. 
In sequencing the SAP-6 proteins and in particular SAP-6-Val human, bovine 
and canine proteins, the following procedure may be utilized. 
Samples of purified SAP-6 proteins are prepared by Edman sequence analysis 
by first dialyzing the samples against water. The samples are taken up in 
methanol and applied to a glass fiber filter. The analysis is performed on 
an Applied BioSystems model 470A Gas Phase Protein Sequencer. Analysis of 
the resulting phenylthiohydantoins (PTH) is accomplished by high pressure 
liquid chromatography (HPLC) at 50.degree. C. on an Altex reversed-phase 
PTH-C.sub.18 column. A binary buffer system consisting of ammonium acetate 
buffered acetonitrile, pH of 4.5, is used. 
The chromatography procedure is conducted utilizing buffer A which contains 
about 10% acetonitrile and buffer B which contains about 90% acetonitrile. 
The initial chromatography conditions are 70% buffer A and 30% buffer B 
with a flowrate of 1.0 ml per minute. One minute after injection, the 
concentration of buffer B is linearly increased to 50% over a 3 minute 
period and held at this level for 8 minutes. The concentration of Buffer B 
then is reduced to 30% over one minute. After re-equilibration, which 
takes approximately 10 minutes, another PTH sample is injected. A 
discussion of this separation procedure appears in greater detail in 
Joseph L. Meuth and J. Lawrence Fox, Separation of Amino Acid 
Phenylthiohydantoin Derivatives by High-Pressure Liquid Chromatography, 
Analytical Biochem, 154(2):478-484, 1986, which is incorporated herein by 
reference in its entirety. 
The sequence for the first 14 amino acid residues after the amino-terminal 
residue for the other human 6,000 dalton SAP-6 protein isolated from human 
tissue origin has also been ascertained in accordance with the above 
described sequencing techniques and is as follows: 
Phe--Pro--Ile--Pro--Leu-- 
Pro--Tyr--Cys--Trp--Leu-- 
Cys--Arg--Ala--Leu. 
As already indicated and suggested by the above human NH.sub.2 -terminal 
sequence, there is believed to exist more than one 6,000 dalton 
hydrophobic surfactant-associated protein. With respect to these SAP-6 
proteins, the following has been observed. First, the N-terminal amino 
acid residue sequences of the SAP-6-Val proteins for human, bovine and 
canine species, although similar to one another as indicated above, appear 
to be substantially different from the other N-terminal amino acid residue 
sequence of the human species set forth immediately above. Secondly, when 
the SAP-6 proteins are subjected to SDS-PAGE gel electrophoresis migration 
in the absence of beta-mercaptoethanol, a sulfhydryl reducing agent, major 
and minor amounts of protein appear. This can be better understood with 
reference to FIG. 1 wherein major amounts of silver stained protein exist 
at the 6,000 and 14,000 molecular weight migratory regions whereas only 
minor amounts of silver stained protein exist at the 20,000 and 26,000 
molecular weight migratory regions. Thirdly, when the SAP-6 proteins are 
subjected to gel electrophoresis migration in the presence of 
beta-mercaptoethanol, silver stained proteins appear only at the 6,000 and 
14,000 molecular weight migratory regions as shown in FIG. 2. In view of 
the above noted observations, it is presently believed that more than one 
SAP-6 protein exists. More particularly, it is thought that two separate 
and distinct SAP-6 proteins exist, i.e., a SAP-6-Val protein and a second 
6,000 dalton SDS-PAGE hydrophobic surfactant-associated SAP-6-protein, and 
it is further believed that they co-elute together, co-purify together via 
SDS-PAGE gel electrophoresis migration, have similar molecular weights of 
about 6,000 daltons as determined in SDS-PAGE, have similar biophysical 
surfactant-like activity, and have similar enzyme resistance. Further, it 
is believed that the larger multimers, i.e., Mr=20,000 and Mr=26,000, of 
one or more of the 6,000 dalton SAP-6 proteins are possibly bonded 
together via sulfhydryl bonds in view of their migration patterns in the 
absence and presence of beta-mercaptoethanol, as illustrated in FIGS. 1 
and 2. Therefore, it should be understood that any 6,000 dalton 
hydrophobic surfactant-associated protein, i.e., a SAP-6 protein, is well 
within the contemplation of this invention. 
The present invention further relates to a novel method for isolating the 
novel SAP-6 proteins. The novel SAP-6 proteins can be purified from, for 
example, animal tissue and fluids, cells, cultivated cells, suitable 
pulmonary surfactant replacement preparations available, such as CLSE or 
Surfactant-TA, and the like. It should be appreciated therefore that the 
expression "animal tissue" as used herein is meant in a broad sense to 
encompass all appropriate sources for the SAP-6 proteins, including but 
not limited to animal tissue and fluid, cells, cultivated cells, pulmonary 
surfactant replacement preparations, such as CLSE and Surfactant-TA, and 
the like. It is preferred, however, to isolate the SAP-6 proteins from 
animal pulmonary tissue or amniotic fluid, and more preferably human 
pulmonary tissue. Solutions containing the novel SAP-6 proteins are 
obtained by extraction of animal tissue in which these proteins occur. For 
instance, after the mammalian lung is excised, the trachea preferably 
should be cannulated and washed repeatedly with several volumes of an 
iced, buffered solution, such as, (pH 7.0-7.4) 0.9% NaCl, to remove 
surfactant from the alveoli. The wash is centrifuged at low speed, for 
example, 1,000.times.g for ten minutes, and should be repeated if 
necessary to remove contaminating blood and white cells or macrophages. 
All procedures preferably should be performed at about 2.degree.-4.degree. 
C. This surfactant material should then be centrifuged at high speed from 
the alveoli wash to sediment particulate materials at about 10,000.times.g 
for about 30 minutes or longer. This pellet preferably should be 
resuspended and washed by repeated centrifugation and sonicated or freeze 
thawed, several times if necessary, in a suitable buffer, such as the one 
above, to uniformly resuspend the materials. 
The resulting white surfactant is pelleted by centrifugation, the aqueous 
material is removed and is extracted with about 10-100 volumes of 
approximately 2:1 ether/ethanol, 3:1 chloroform/methanol or other suitable 
organic solvents at about -30.degree. C. to produce a supernatant 
containing the SAP-6 proteins. Extractions should proceed over night. The 
material should then be centrifuged again at about 10,000.times.g for 
about 20 minutes or longer. The resulting liquid fraction will contain 
lipid and include the novel, SAP-6 proteins of this invention. The liquid 
fraction thereafter can be dried (concentrated) with, for instance, 
N.sub.2 stream, resuspended in an organic solvent, such as chloroform or 
other suitable organic solvents, and applied to a silicic acid column or 
LH-20 column, approximately 50 cm.times.2.5 cm in size which is 
pre-equilibrated with chloroform or other suitable organic solvents at 
room temperature. The column is then eluted with, for example, 50 to 200 
mls of a mixture of chloroform and alcohol, such as methanol or the like, 
increasing the alcohol concentration from 0% to about 100% in stepwise or 
gradient fashion. The SAP-6 proteins are eluted in the solvents containing 
alcohol having concentrations of greater than about 40% up to about 100% 
and more particularly about 40% to about 80%. Most of the lipids present 
in the SAP-6 proteins will be separated from the proteins at this stage. 
It should be appreciated to those skilled in the art that the silicic acid 
column can also be eluted with any other suitable solvents capable of 
extracting therefrom the hydrophobic SAP-6 proteins of this invention. 
Fractions containing mixtures of the SAP-6 proteins of this invention now 
can be assessed by, for example, fluorescamine assay and gel 
electrophoresis migration (SDS-PAGE or tricine-SDS-PAGE) followed by 
staining with silver stain or Coomassie staining. The SAP-6 proteins 
isolated at this point is considered to be substantially pure and 
homogeneous. By "substantially pure" it is meant herein that the SAP-6 
proteins present therein are substantially free of contaminants, such as 
cells, cellular debris, DNA, RNA, and major surfactant glycoproteins or 
fragments thereof. It should be appreciated, however, that some lipids 
still remain in the now substantially pure protein factor. 
To produce virtually pure SAP-6-Val protein, the fractions containing the 
mixtures of eluted SAP-6 proteins preferably are pooled, evaporated to 
near dryness and subjected to repeated dialysis in, for example, a 
cellulose dialysis bag with about 100-500 volumes of about 2:1 of 
chloroform/methanol or other suitable mixtures or acidified 
chloroform/methanol mixtures acidified with HCI or other appropriate acids 
at about room temperature. This procedure should proceed until the 
SAP-6-Val protein is virtually free of contaminating lipids and other 
SAP-6 proteins. This substantially purified SAP-6-Val protein can also be 
assessed by fluorescamine assay and gel electrophoresis migration. It 
should be realized that other suitable delipidating steps may also be 
employed to accomplish the above delipidating objective. The approximate 
amino acid composition can now be determined and the SAP-6-Val protein can 
be identified after PAGE with Coomassie, silver staining or other standard 
staining techniques. It should be appreciated that the above methods are 
described in generality and can be utilized with other suitable solvents 
and methods to separate the novel SAP-6 proteins from appropriate sources, 
and to further separate the novel SAP-6-Val protein from the SAP-6 
proteins. 
It should now be well apparent to those versed in the art that SAP-6 
proteins and the SAP-6-Val protein can be isolated from several animals 
and in particular human animals and, once sequenced, can be synthesized by 
direct peptide synthesis or by genetic engineering technology to provide 
adequate synthetic material required for medicinal and research use. A 
discussion of the SAP-6 proteins appears in Whitsett et al, Hydrophobic 
Surfactant-Associated Protein (SAP 6-14). In Whole Lung Surfactant and Its 
Importance for Biophysical Activity in Lung Surfactant Extracts Used for 
Replacement Therapy, Pediatr Res, 20(5):460-467, 1986, and Whitsett et al, 
Immunologic Identification of a Pulmonary Surfactant-Associated Protein of 
Mr=6,000 Daltons, Pediatr Res, 20(8):744-749, 1986, in which these 
articles are incorporated herein by reference in their entireties. 
It is believed that the SAP-6 proteins of this invention have useful 
"surfactant-like activity". As used herein, "surfactant-like activity" 
refers to the ability to interact with phospholipids to reduce surface 
tension spread on a surface and/or reduce surface tension at an air/liquid 
interface. When the SAP-6 proteins and in particular the purified 
SAP-6-Val proteins are mixed with phospholipids, their unique ability to 
enhance the surfactant-like activity of phospholipids in lungs of an 
animal is believed to impart to the mixture significant pulmonary 
biophysical surfactant activity. Such a mixture with this activity is 
highly useful for replacing or supplementing natural pulmonary surfactant 
material for reducing or maintaining normal surface tension in lungs, 
especially in the lungs of animals suffering from HMD and other syndromes 
associated with the lack or insufficient amounts of natural pulmonary 
surfactant material. 
To prepare a preparation for medicinal application comprising a mixture of 
the SAP-6 proteins or purified SAP-6-Val proteins and phospholipids, a 
mixture of isolated SAP-6 proteins or purified SAP-6-Val proteins can be 
stored in chloroform under nitrogen or air at about -30.degree. C. prior 
to reconstitution with phospholipids. The phospholipids that may be 
employed can be derived from a number of suitable phospholipids, such as 
phosphatidylcholine, disaturated phosphatidylcholine, 
phosphatidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositol 
and mixtures thereof and the like. For example, isolated SAP-6 proteins or 
purified SAP-6-Val proteins can be mixed in an amount of about 0.1 to 
about 2.0% with synthetic phospholipids, such as about 65% 
dipalmitoylphosphatidycholine, about 20% egg-phosphatidycholine, about 
7.5% of soy-phosphatidylinositol, and about 7.5% egg-phosphatidylglycerol. 
If desired, in preparing such a medicament, the mixture may further 
contain a physiological buffer comprising 0.9% sodium chloride with or 
without about 0.5 to about 1.5 mM calcium chloride or any other suitable 
pharmaceutically accepted carrier. After reconstitution or mixing, the 
material should be sonicated or vortexed and tested for surface tension 
lowering capacity and adsorption on a Wilhelmy balance or other surface 
tension measuring devices. Adsorption studies can be conducted by the 
methodology disclosed by Notter et al, Chem. Phys. Lipids, 33:67-80, 1983 
and Ross et al, Phospholipid Binding and Biophysical Activity of Pulmonary 
Surfactant-Associated Protein (SAP)-35 and Its Non-Collagenous 
COOH-terminal Domains, 261(30):14283-14291, 1986, in which they are 
incorporated herein by reference in their entireties. 
A phospholipid mixture (SM) containing about 65% 
dipalmithoylphosphatidylcholine (DPPC), about 20% egg-phosphatidycholine 
(PC), about 7.5% egg-phosphatidyglycerol (P6) and about 7.5% 
soy-phosphatidylinositol (PI) is combined with SAP-6-Val proteins and 
tested in a Wilhelmy balance and nitrogen bubble surfactometer. The 
SAP-6-Val protein is adsorbed rapidly, lowers surface tension to about 
30-47 dynes/cm and is found to be more active than the phospholipid 
mixture or SAP-35, the major surfactant-associated glycoprotein, mixed 
with the same phospholipid mixture tested in identical fashion. See Tables 
II-IV. The methods employed to generate Tables II-IV can be found in 
detail in Notter et al and Ross et al referenced hereinabove. 
TABLE II 
__________________________________________________________________________ 
ADSORPTION FACILITY OF A SYNTHETIC LIPID MIXTURE (SM)*, 
A SYNTHETIC LIPID MIXTURE (SM) COMBINED WITH SAP-35, 
AND A SYNTHETIC LIPID MIXTURE (SM) COMBINED 
WITH SAP-6 PROTEINS 
Lipid 
Concentration 
Adsorption Surface Pressure .pi. at time 
Mixture (mg lipid/ml subphase) 
t = 0 min 
5 min 
10 min 
15 min 
__________________________________________________________________________ 
SM 0.6 mg/ml 1 dyne/cm 
1 1 3 
SM + SAP-35** 
0.6 mg/ml 20 dynes/cm 
22 22 22 
(canine) 
SM + SAP-6** 
0.6 mg/ml 43 dynes/cm 
45 45 45 
Proteins (human) 
__________________________________________________________________________ 
*SM is a synthetic lipid mixture composed of DPPC:eggPC:egg-PG:soy-PI 
65:20:7.5:7.5. 
**SAP35 and SAP6 proteins have been added in similar molar ratios. 
TABLE III 
______________________________________ 
Adsorption of Delipidated SAP-35 and Various Segments and 
SAP-6-Val Combined with a Synthetic Phospholipid Mixture 
**Surface Pressure .pi. (dynes/cm) 
Surfactant 0 30 
Mixture* min 5 min 10 min 
15 min 
20 min 
min 
______________________________________ 
1. SM 1 1 1 3 3 
2. SM + 1 2 4 5 5 
Ovalbumin 
3. SM + 9 13 14 15 18 
SAP-35 
4. SM + 8 16 17 17 18 
SAP-18 
5. SM + 2 4 6 7 9 
SAP-21 
6. Natural LS 17 46 46 47 47 
7. SM + 19 26 29 31 33 37 
Canine 
SAP-6-Val 
(25 .mu.g) 
8. SM + 37 47-48 47-48 47-48 47-48 
Canine 
SAP-6-Val 
(50 .mu.g) 
9. SM + 46 47 47 47 47 
Canine 
SAP-6-Val 
(100 .mu.g) 
______________________________________ 
*Delipidated proteins studied are glycoproteins SAP35, SAP21 and SAP18 an 
canine hydrophobic protein SAP6-Val. Each of these proteins are combined 
with a synthetic phospholipid mixture (SM) composed of 
DPPC:eggPC:egg-PG:soy-PI 65:20:7.5:7.5. Natural LS (lipidsurfactant) 
represents optimally active surfactants; ovalbumin is used as a 
nonspecific protein control. Phospholipid concentration for all adsorptio 
experiments is about 0.063 mg/ml 0.15 M NaCl with about 1.4 mM CaCl.sub.2 
; temperature = 35 .+-. 2.degree. C. For cases 2-5, the lipid to protein 
ratio is (wt/wt) 99/1 to 98/2. All mixtures are dispersed by vortexing at 
room temperature except natural LS (resuspended). 
**Surface pressure .pi. is the amount of surface tension lowering below 
that of the pure subphase. The .pi. value at 0 min. is that measured 
within 10 seconds after addition of a bolus of surfactant dispersion to 
the stirred subphase at time zero. Values given for 1-6 are the means of 
4-10 experiments with SEM (standard error of measure) always less than 3 
dynes/cm, except for SAP18 data which are the mean of 2 experiments with 
deviation of .+-. 0.5 dynes/cm about the me# an. 
TABLE IV 
__________________________________________________________________________ 
Dynamic Surface Tension Lowering During Dynamic 
Compression on an Oscillating Bubble 
Minimum Surface 
.pi. After Cycling 
Surfactant Dispersion Conc. 
Tension (dynes/cm) 
For 
Dispersion* (mg lipid/ml) 
0.5 min 
2 min 
5 min 
10 min 
11 min 
__________________________________________________________________________ 
SM 1 mg/ml 55 21 21 21 
Egg-PC 1 mg/ml 53 42 27 22 
SM + SAP-35 
1 mg/ml 36 36 36 36 
SM + SAP-35 
2 mg/ml 35 23 21 21 
SM + SAP-21 
1 mg/ml 54 47 47 42 
LS 1 mg/ml 16 10 2 1 
SM + Canine 
1 mg/ml 15 15 14 3 1 
SAP-6-Val (25 .mu.g) 
SM + Canine 
.5 mg/ml 17 16 16 16 16 
SAP-6-Val (25 .mu.g) 
SM + Canine 
2 mg/ml 16 9 4 3 1 
SAP-6-Val (25 .mu.g) 
10. 
SM + Canine 
.5 mg/ml 16 15 1 1 etc. 
SAP-6-Val (50 .mu.g) 
SM + Canine 
1 mg/ml 15 0 0 0 etc. 
SAP-6-Val (50 .mu.g) 
SM + Canine 
2 mg/ml 15 13 1 etc. 
etc. 
SAP-6-Val (50 .mu.g) 
__________________________________________________________________________ 
*Surfactant dispersions are studied on the oscillating bubble at about 
37.degree. C., about 100% humidity, cycling rate about 20 cpm and area 
compression about 50%. The concentration is 1 or 2 mg lipid/ml bubble 
subphase as noted. Natural LS (lipidsurfactant) represents active lung 
surfactant preparation against which to compare the lipidprotein combined 
mixtures of cases 3-5 and 7-10. 
In treating HMD and other related disorders, mixtures containing SAP-6 
proteins mixed with naturally occurring or synthetic phospholipids can be 
instilled intratracheally, for instance, in liquid form or as an aerosol 
spray in doses of about 0.1% to about 2.0%, by weight, SAP-6 proteins with 
about 20 to about 100 mg phospholipid per kilogram for treatment or 
prevention of HMD and other related disorders. 
Another utility for SAP-6 proteins is in the preparation of antibodies or 
antisera against them. Immunochemical methods can be used for detecting 
and determining SAP-6 proteins, for example, in a fraction from a 
separation operation, since SAP-6 proteins have antigenic properties. 
Immunoblot and ELISA analysis can be used for immunological detection and 
quantification or other immunological assays of SAP-6 proteins. 
An antiserum which can be used for this purpose can be obtained as follows: 
antisera with which purified SAP-6 proteins can be detected can be 
obtained by immunization of albino rabbits with repeated injections of 
delipidated purified SAP-6 proteins and Freund's complete adjuvant. Usable 
antisera should be obtainable after four injections with approximately 100 
micrograms of the delipidated SAP-6 proteins per injection. The antigen 
used should contain purified SAP-6 proteins assessed by silver staining 
analysis after one dimension SDS-PAGE. Hybridoma monoclonal antibodies and 
polyclonal antibodies can also be produced and used. 
The detection and determination of SAP-6 proteins by immunological methods 
is of diagnostic importance. In normal situations, since SAP-6 proteins 
occur in some tissues of the human body, for example, lung tissue, lung 
lavage and amniotic fluid, SAP-6 proteins can be detected. In illnesses 
associated with SAP-6 proteins, the proteins will not appear or will 
appear in altered concentrations in the serum, tissue, such as the lungs, 
or other body fluids. The detection or lack of detection of these proteins 
can be useful for diagnostic determination of an illness or for monitoring 
the course of a disease and for closely controlling the therapy of such an 
illness. SAP-6 proteins have been detected in human amniotic fluid near 
term gestation by immunoblot analysis using anti-SAP-6 protein antisera. 
SAP-6 proteins can thus be used to prepare antisera and/or antibodies 
which can be employed to detect and determine SAP-6 proteins. 
With respect to the production of antibodies against SAP-6 proteins, 
standard known techniques for producing such antibodies are certainly 
within the contemplation of this invention. As one example, lymphocytes 
from mice immunized with isolated SAP-6 proteins may be fused with mouse 
myeloma cells in the presence of, for example, polyethylene glycol, 
generally according to the technique of Kohler and Milstein, 256:495-497 
(1975), which is incorporated herein by reference in its entirety. The 
surviving hybrids would then be screened by selective cloning to obtain 
only cells which produce the anti-SAP-6 antibody substance. The cell 
line(s) so obtained would be grown in suspension culture to provide 
substantial quantities of anti-SAP-6 which, when separated from 
contaminating myeloma antibody by immuno-absorption, may be employed in 
immunoassay techniques for detection of SAP-6 proteins in, for instance, a 
tissue sample. For example, such antibodies can bind to or precipitate 
SAP-6 proteins from a mixture containing SAP-6 proteins, increase the 
sedimentation rate of SAP-6 proteins, can modify elution characteristics 
of SAP-6 proteins on gel filtration, and can identify or quantify SAP-6 
proteins from a sample. 
A mixture of isolated SAP-6 proteins and purified SAP-6-Val proteins are 
obtained in accordance with the instant invention as recited in the 
following example: 
EXAMPLE 
I. Isolating Surfactant Material 
Surfactant-associated proteins are isolated from lung lavage material which 
is obtained from adult dogs and cows after sacrifice. Proteins are also 
isolated from human surfactant which are obtained from human cadavers 
after autopsy or from lung lavage of consenting adults. In animal studies, 
the trachea is cannulated and the lung is lavaged three times with several 
volumes of iced 0.9% NaCl, 50 mM Na.sub.2 HPO.sub.4, and 5 mM EDTA, pH 
7.2. Cells and debris are removed by centrifugation at 80.times.g for 
about 10 minutes (twice) and a particular fraction is collected by 
centrifugation at 40,000.times.g for about 30 minutes at 4.degree. C. The 
pelleted material is then resuspended in the above buffer containing 1 mM 
phenylmethylsulfonylfluoride and sonicated for 10 seconds with a Branson 
sonifier. Surfactant is pelleted by repeated centrifugation at about 
40,000.times.g for about 30 minutes at 4.degree. C. 
II. Purification of a Mixture of SAP-6 
Proteins From Surfactant Material 
The surfactant pellet is further processed by extraction in about 3:1 
ether/ethanol or chloroform/methanol at about -30.degree. C. for about 16 
hours. Ether/ethanol or chloroform/methanol extracts containing SAP-6 
proteins are evaporated to near dryness with N.sub.2 gas and are 
redissolved in chloroform. The redissolved residue in chloroform is 
applied to a BioSil-HA column (silicic column dimensioned 2.5 cm by 40 cm 
that is obtained from Bio-Rad, Richmond, Calif.) and is equilibrated in 
chloroform. Lipid-protein fractions are recovered by stepwise elution with 
chloroform-methanol mixtures (200 ml each) with successive increases in 
concentration of methanol (10% intervals) in chloroform. The SAP-6 
proteins are eluted between 3:2 and 1:4 chloroform/methanol solvent 
elution or about 40% to about 80% methanol in chloroform. 
III. Purification of SAP-6 
-Val Proteins From A Mixture of the SAP-6 
Proteins 
To obtain virtually pure SAP-6-Val protein, the SAP-6 proteins are 
subjected to repeated dialysis in a cellulose dialysis bag with about 
100-500 volumes of about 2:1 of chloroform/methanol at room temperature. 
The present invention may, of course, be carried out in other specific ways 
than those herein set forth without departing from the spirit and 
essential characteristics of the invention. The present embodiments are, 
therefore, to be considered in all respects as illustrative and not 
restrictive and all changes coming within the meaning and equivalency 
range of the appended claims are intended to be embraced herein.