Modified serum albumin with reduced affinity for nickel and copper

#This invention relates in general to a modified serum albumin useful as a blood volume expander, and in particular to a recombinant or otherwise modified serum albumin that is mutated such as by truncation, elongation or insertion at its n-terminal region which has improvements in its metal binding properties and reduced affinity for metal elements such as nickel or copper and which can thus be used safely and effectively in a variety of applications including use as a blood volume expander, for excipient and culture media applications, or as additives or substitutes in a wide range of products including medicines, cosmetics, dairy products and dietary supplements which would normally utilize unmodified forms of albumin or related compounds.

#The serum albumins belong to a multigene family of proteins that includes alpha-fetoprotein and human group-specific component, also known as vitamin-D binding protein. The members of this multigene family are typically comprised of relatively large multi-domain proteins, and the serum albumins are the major soluble proteins of the circulatory system and contribute to many vital physiological processes. Serum albumin generally comprises about 50% of the total blood component by dry weight, and as such is responsible for roughly 80% of the maintenance of colloid osmotic blood pressure and is chiefly responsible for controlling the physiological pH of blood.

#The albumins and their related blood proteins also play an extremely important role in the transport, distribution and metabolism of many endogenous and exogenous ligands in the human body, including a variety of chemically diverse molecules including fatty acids, amino acids, steroids, calcium, metals such as copper and zinc, and various pharmaceutical agents. The albumin family of molecules are generally thought to facilitate transfer many of these ligands across organ-circulatory interfaces such as the liver, intestines, kidneys and the brain, and studies have suggested the existence of an albumin cell surface receptor. See, e.g., Schnitzer et al., P.N.A.S. 85:6773 (1988). The albumins are thus intimately involved in a wide range of circulatory and metabolic functions.

#The present invention will now be described in detail with respect to preferred embodiments thereof, which are to be taken together with the accompanying drawings, wherein:

#FIG. 1 is a view of the structure of human serum albumin illustrating the binding region VI as determined crystallographically.

#FIG. 2 is a photographic view of a serum albumin which has been mutated in accordance with the present invention (left side) and which is virtually free of trace metals as compared to a non-modified serum albumin (right side) which retains far greater trace metals such as nickel and copper than the modified albumin of the present invention.

#In accordance with the present invention, a modified human serum albumin is provided which is modified at the binding site VI, or the n-terminal region, so as to have reduced or eliminated affinity to trace metals such as nickel and/or copper. A schematic crystallographic view of a human serum albumin crystal showing some of the albumin binding regions including binding region VI is shown in FIG. 1. By human serum albumin, it will be understood that this refers to the human form of the serum albumin protein, or any other serum albumin protein that operates in a similar manner to the human serum albumin and has binding sites with similar properties. Accordingly, any serum albumin that has sufficient similarity with regard to the properties of binding region VI, e.g., other serum albumins including bovine serum albumin (BSA), are contemplated as useful in accordance with the invention. In addition, it will also be the case that other peptides, fragments, or other subunits that contain the binding region VI of human serum albumin or which are equivalent to this region and have the same properties will also fall within the scope of the invention since these fragments or subunits may be isolated and perform in the same manner as an natural albumin which contains this binding region. These peptides, fragments or other subunits of albumin which contain the binding region VI thus can also be modified in the manner of the invention as set forth herein so as to reduce or eliminate unwanted binding of trace metals such as nickel or copper in those applications wherein such reduction or elimination is desirable. Binding region VI in the human serum albumin is located at the n-terminal region and which includes residues 1–3 of the amino acid chain, and has the sequence Asp-Ala-His.

#In accordance with one of the preferred embodiments of the present invention, a modification which can achieve the desired reduction of affinity is made by truncating the serum albumin at the n-terminal end by at least one amino acid so that the affinity to trace metals is reduced. In the particularly preferred embodiment, the truncation will comprise the removal of a single amino acid from the n-terminal end of the serum albumin which is sufficient to achieve reduction or elimination of trace metal binding, but will not have any other appreciable effects on the albumin and its normal properties. However, although a truncation of a single amino acid will be sufficient to accomplish the reduction in metal affinity, truncations of more than one amino acid from the n-terminal end will also accomplish the purpose of lowering the albumin's affinity to trace metals.

#Similarly, other modifications which alter the n-terminal end of the albumin will also be suitable in reducing or eliminating the affinity to undesirable trace metals. For example, such mutations may take the form of elongation or insertion of suitable amino acids so as to change the character of the n-terminal region and make the binding site region VI far less likely to bind and/or retain trace metals. Any elongation or insertion at the n-terminal region or within binding region VI that is sufficient to either sterically hinder the binding site VI or eliminate vital binding interactions in this region will be suitable for use in the present invention and will provide the interference necessary to inhibit or eliminate the binding of the albumin with trace metals including nickel and copper. Although a variety of amino acid sequences of differing sizes will be useful in accordance with the invention, elongations or insertions of peptides having from about 1 to 10 amino acids are generally preferred.